2004 CROH Cancer stem cells and differentiation therapy.

Critical Reviews in Oncology/Hematology51(2004)1–28

Stem cell origin of cancer and differentiation therapy

Stewart Sell?

Center and Ordway Research Institute,Empire State Plaza,New York State Health Department,Wadsworth Center,

P.O.Box509,Room C-400,Empire State Plaza,Albany,NY12201,USA

Accepted8April2004

Contents

Abstract (2)

1.Stem cells (2)

1.1.Embryonal,germinal and somatic(adult)stem cells (2)

1.2.Symmetric and asymmetric division (3)

1.3.Stem cell signals,development and cancer (4)

1.4.Potential/plasticity of stem cells (5)

1.5.Adult stem cells and normal tissue renewal (6)

1.6.Pleopotency of bone marrow stem cells (7)

2.Stem cells and cancer (7)

2.1.Embryonal rest hypothesis (7)

2.2.Anaplasia (8)

2.3.Maturation arrest (8)

3.Childhood tumors (8)

3.1.Wilms tumor (8)

3.2.Neuroblastomas (8)

4.Germinal stem cells and teratocarcinoma (9)

4.1.Totipotent embryonal cancer stem cells (9)

4.2.Teratocarcinomas (9)

4.3.Control of malignant potential of embryonal cancer cells (9)

4.4.Embryonic cells compared to embryonal carcinoma cells (10)

4.5.Regulation of tumor cell differentiation (10)

5.Skin cancer (10)

5.1.Skin cell lineage and malignant phenotype (10)

5.2.Skin cancers arise in self-renewing stem cells(initiation and promotion) (10)

5.3.Field cancerization (11)

6.Liver cancer (12)

6.1.Participation of various cells in the hepatocyte lineage in hepatocarcinogenesis (12)

6.2.Fusion of bone marrow and liver cells (13)

7.Adenocarcinomas (13)

7.1.Stem cell origin (13)

8.Hematopoietic stem cells and leukemia (13)

8.1.Normal blood renewal (13)

8.2.Multi/pluripotent hematopoietic stem cells (13)

8.3.Bone marrow transplantation and pleotypic potential (14)

8.4.Potentiality caveats (14)

8.5.Unanswered questions (15)

9.Leukemias as models of maturation arrest of a cell lineage (15)

9.1.Leukemia as a stem cell cancer (15)

9.2.Chemotherapy and reserve stem cells (15)

?Tel.:+15184740547;fax:+15184732900/4025381.

E-mail address:ssell@https://www.wendangku.net/doc/9f15720943.html,(S.Sell).

1040-8428/$–see front matter?2004Elsevier Ireland Ltd.All rights reserved.

doi:10.1016/j.critrevonc.2004.04.007

2S.Sell/Critical Reviews in Oncology/Hematology51(2004)1–28

9.3.Gene translocations and leukemias (16)

9.3.1.The IL-3receptor (16)

9.3.2.B-cell lymphomas and the immunoglobulin heavy chain gene promoter (17)

9.4.Stage of maturation arrest and clinical course of leukemias (17)

10.Differentiation therapy (18)

10.1.Retinoic acids and teratocarcinomas (18)

10.2.Retinoids and acute promyelocytic leukemia(APL) (19)

10.3.Retinoid differentiation therapy;other cancers (20)

10.4.Chemoprevention of cancer by retinoids (20)

10.5.Retinoic acid syndrome (20)

11.Summary and conclusions (20)

Reviewers (21)

Acknowledgements (21)

References (21)

Biography (28)

Abstract

Our forefathers in pathology,on observing cancer tissue under the microscope in the mid-19th century,noticed the similarity between embryonic tissue and cancer,and suggested that tumors arise from embryo-like cells[1,2][Recherches dur le Traitement du Cancer,etc.Paris. (1829);Editoral Archiv fuer pathologische Anatomie und Physiologie und fuer klinische Medizin8(1855)23].The concept that adult tissues contain embryonic remnants that generally lie dormant,but that could be activated to become cancer was later formalized by Cohnheim [3,4][Path.Anat.Physiol.Klin.Med.40(1867)1–79;Virchows Arch.65(1875)64]and Durante[5][Arch.Memori ed Osservazioni di Chirugia Practica11(1874)217–226],as the“embryonal rest”theory of cancer.An updated version of the embryonal rest theory of cancer is that cancers arise from tissue stem cells in adults.Analysis of the cellular origin of carcinomas of different organs indicates that there is,in each instance,a determined stem cell required for normal tissue renewal that is the most likely cell of origin of carcinomas[6] [Lab.Investig.70(1994)6–22].In the present review,the nature of normal stem cells(embryonal,germinal and somatic)is presented and their relationships to cancer are further expanded.Cell signaling pathways shared by embryonic cells and cancer cells suggest a possible link between embryonic cells and cancer cells.Wilm’s tumors(nephroblastomas)and neuroblastomas are presented as possible tumors of embryonic rests in children.Teratocarcinoma is used as the classic example of the totipotent cancer stem cell which can be in?uenced by its environment to differentiate into a mature adult cell.The observation that“promotion”of an epidermal cancer may be accomplished months or even years after the initial exposure to carcinogen(“initiation”),implies that the original carcinogenic event occurs in a long-lived epithelial stem cell population.The cellular events during hepatocarcinogenesis illustrate that cancers may arise from cells at various stages of differentiation in the hepatocyte lineage.Examples of genetic mutations in epithelial and hematopoietic cancers show how speci?c alterations in gene expression may be manifested as maturation arrest of a cell lineage at a speci?c stage of differentation.Understanding the signals that control normal development may eventually lead us to insights in treating cancer by inducing its differentiation(differentiation therapy). Retinoid acid(RA)induced differentiation therapy has acquired a therapeutic niche in treatment of acute promyelocytic leukemia and the ability of RA to prevent cancer is currently under examination.

?2004Elsevier Ireland Ltd.All rights reserved.

Keywords:Stem cells;Cancer;Differentiation;Plasticity;Carcinogenesis;Embryonal rests;Differentiation therapy;Retinoic acid

“The simplest view appears to me undoubtedly to be that in an early stage of embryonic development more cells are produced than are required for building up the part concerned,so that there remains unappropriated a quantity of cells--it may be very few in number--which,owing to their embryonic character,are endowed with a marked capacity for proliferation...The only point on which I lay stress is that the real cause of the subsequent tumour is to be sought in a fault or irregularity of the embryonic rudiment.”Julius Cohnheim,1889

“Oncogeny is blocked ontogeny.”Van R.Potter,1968“Cancer is a problem of developmental biology.”G.Barry Pierce et al.19781.Stem cells

1.1.Embryonal,germinal and somatic(adult)stem cells There are three kinds of stem cells:embryonal,germinal, and somatic or adult stem cells[7].Embryonal stem cells are derived from the?rst?ve or six divisions of the fertilized egg.The progeny of embryonal stem cells are the precursors for all of the cells of the adult organs.Germinal stem cells in the adult produce eggs and sperm and are responsible for reproduction.Somatic stem or progenitor cells are consid-ered more limited in their potential,and they produce cells that differentiate into mature functioning cells and that are responsible for normal tissue renewal.The ultimate stem cell,the fertilized egg and its embryonic stem cell progeny

S.Sell/Critical Reviews in Oncology/Hematology51(2004)1–28

3

Fig.1.Stem cell lineages in adult tissues and normal tissue renewal.In analogy to the structure of a tree with a very small trunk,normal tissue stem cells represent a very small proportion of the tissue cells and do not normally proliferate.The differentiating progeny of the tissue-determined stem cells is represented by the branches,which represent lineages of cells eventually giving rise to the terminally differentiated functional parenchymal cells, represented by the leaves.For deciduous trees,loss of the leaves at the end of the year(falling off or apoptosis)occurs in cycles,whereas in animal tissues there is a continuous production and loss of cells.Cancer results when the rate of proliferation increases and the rate of cell loss remains the same or when the rate of proliferation stays the same and/or the rate of loss decreases.

are totipotent and give rise to branches(lines)of cells that form various differentiated organs.During this process,the progeny of the embryonic stem cells lose potential and gain differentiated properties,in a poorly understood process called determination.In the adult,the cells responsible for tissue renewal are no longer totipotent,but become more re-stricted in their ability to form different tissues.An exception to this rule may be germinal cells.Some tumors of germinal cells(embryonal carcinomas)are able to produce differenti-ated cell types of all adult organs,as well as placental tissues (see below).It is generally accepted that adult tissues contain tissue-determined stem cells responsible for normal tissue renewal.However,there is increasing evidence for retention of some stem cells in the non-germinal tissues of adults,es-pecially in the bone marrow,that retain the potential to pro-duce different cell lineages.These“reserve”stem cells are very few in number as most of the cellular renewal is accom-plished by tissue determined transit-amplifying cells(Fig.1).

1.2.Symmetric and asymmetric division

The various types of stem cells not only have different potentials,but they also proliferate differently.Cells can di-vide symmetrically,whereby each daughter cell retains the properties of the parental cells,or asymmetrically,whereby one daughter cell retains the properties of the parental stem cell,whereas the other daughter cell begins the process of determination(Fig.2)[8].The characteristic of embryonal stem cells is that they divide symmetrically.Each daughter cell remains a totipotent stem cell,resulting in a logarithmic expansion of cells during early embryonic growth.Then,as the germ layers of the early embryo form and the process of determination begins,the cells proliferate asymmetrically. The tissue determined stem(progenitor cells)divide asym-metrically as one daughter cell remains to continue the pro-cess of cell renewal and the other daughter cell starts the process of differentiation(transit amplifying cells).The de-termined transit amplifying cells retain the ability to divide for several differentiation stages and are the major contribu-tors to normal tissue renewal.These cells are known as pro-genitor cells in the bone marrow,and as transit-amplifying cells in the skin and other tissues.In these and other rapidly proliferating tissues the stem cells are normally quiescent and do not divide.The tissue is renewed by proliferation of the daughter cells(transit amplifying cells),which con-tinue the process of determination.Finally,the end product is a terminally differentiated cell(red blood cell or poly-morphonuclear cell in the blood;keratinized epithelial cell in the skin).

The molecular mechanism of asynchronous division is poorly understood.In1975,Cairns hypothesized that the reserve tissue stem cell retains an immortal reserve DNA template which is passed on each division to the reserve stem cells,whereas the transit amplifying cells receive only newly synthesized DNA[9].Although this is dif?cult to prove,there are recent data that this may occur in a tis-sue culture of a mouse?broblast cell line[10],as well as in the crypts of the small intestine[11].How this re-

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1–28

Fig.2.Symmetric and asymmetric division.When cells divide symmetrically,each daughter cell is identical and retains the potential of the parental cell.When cells divide asymmetrically,one of the daughter cells remains a stem cell,whereas the other begins the process of determination [310,311].In adult tissues,the progeny of the tissue stem cells undergoing differentiation (determination)retain the capacity to proliferate (transit-amplifying cells)and comprise the major source of cells for normal tissue renewal.

serve DNA functions to maintain the tissue stem cell is not known.

Asynchronous division may be determined by anatomical boundries.In germinal cells,one pole of an asymmetrically dividing cell contains different protein complexes than are present at the other pole [12,13]or one of the poles may be anchored to a supporting cell [14]or membrane by a cad-herin,?-catenin,APC (adenomatous polyposis coli)com-plex (see below),so that one daughter cell remains attached to a surrounding cell whereas the other does not and begins the process of determination [15].It appears that asymmet-ric division of adult stem cells can be suppressed by the purine nucleoside xanthosine,which stimulates guanine ri-bonucleotide synthesis and promotes symmetric clonal ex-pansion.This is reported to be a mechanism that can be used to derive liver stem cell lines from adult tissues [16].1.3.Stem cell signals,development and cancer

Understanding what controls the maintenance of stem cells and differentiation signals may give insights into the cellular signals involved in cancer,and may ultimately lead to new approaches to differentiation therapy.The signal that controls which daughter cell of an adult stem cell remains a stem cell and which begins the process of determination may be mediated through a number of signaling pathways including the Oct-4,Wnt/?-catenin,Notch,BMP (bone mor-phogenic protein),Janus family kinase,or sonic hedgehog signaling pathways,etc.[13,17–19].Two of these,Oct-4and the Wnt/?-catenin are presented as examples.

Oct-4is a transcription factor that is expressed only in the inner cell mass of the embryo,but not in the trophecto-derm,the structure that will form the extra-embryonic tis-sues.During later development Oct-4expression is restricted to cells of the germ line [20],and is essential in maintaining totipotency and synchronous division.Loss of expression is associated with differentiation of cells.The human papillo-mavirus E7oncoprotein speci?cally binds to a domain on the Oct-4promoter to activate Oct-4expression leading to viral transformation [21].Oct-4is a marker for embryonal cancer [22],and treatment with retinoids,which induce dif-ferentiation of embryonal carcinomas,causes a decrease in expression of Oct-4[23].Thus,Oct-4expression appears to be important in maintaining the undifferentiated state of embryonal carcinoma [24],as well as in other cancers.Un-derstanding how to block Oct-4promises to be an approach to controlling the differentiation state of some of the least differentiated cancers (differentiation therapy,see below).Other pathways are important at later stages of differenti-ation and activation of the genes controlling these pathways could act later in adult tissues as oncogenes [19],hold-ing cells in a state of maturation arrest.For example,the Wnt/?-catenin cell activation pathway (Fig.3)is active in maintaining proliferation at an early stage of differentiation and may have a similar role in cancer cells [18,25].Wnt signaling affects the orientation of the chromosomes during mitotic division,and abnormalities in the orientation might contribute to mitotic disjunctions typical of cancer cells [26].In general,Wnt/?-catenin signaling activates proliferation [27]and inhibits apoptosis [28,29],the classic hallmarks of cancer cells.However,this pathway may also be instru-mental for differentiation signals critical in determining the fate of cells during development of many different organs [17,30–34].Cell proliferation and differentiation in the

S.Sell/Critical Reviews in Oncology/Hematology51(2004)1–285

Fig.3.The Wnt/?-catenin signaling pathway.When this signaling path-way is not activated,?-catenin is bound to adherins in the junctions between cells,and unbound?-catenin is tagged for destruction by a complex of proteins including the adenomatous polyposis coli protein (APC).Activation of the Wnt signaling pathway by binding of Wnt to the seven-pass transmembrane Frizzled receptor leads to phosphorylation of the disheveled protein(Dsh),which then associates with axin and pre-vents glycogen synthase kinase3(Gsk-3)from phosphorylating critical substrates,the negative regulators axin and APC,as well as?-catenin itself[26].The unphosphorylated cellular?-catenin escapes recognition by?-TRCP,a component of an E2ubiquitin ligase,and is protected from ubiquinization and destruction.The cellular?-catenin then translocates to the nucleus,where it associates with transcription factors,such as Tcf and Lcf[43,312],and activates transcription of cell proliferation genes,in particular,cyclin D1,c-jun and c-myc[41,51].Modi?ed from[26,313]. wing of the?y Drosophila is controlled by region-speci?c signals that act through the Wnt pathway[35].Long-term hematopoietic stem cells show up-regulation of the frizzled Wnt receptor[36].Thus,Wnt/?-catenin signaling appears to be at least one of the critical means by which prolif-eration and differentiation of cells are controlled during development,and it could be predicted that this pathway also plays a critical role in cancer.

Wnt was recognized as a putative oncogene over20 years ago,before it was found to be the homologue of the Drosophila wingless(wnt-1)gene[37].Transfection of tis-sue culture cells,with the proto-oncogene c-wnt,results in accumulation of cellular?-catenin and morphologic trans-formation[38,39].Expression of a truncated,constitutively active form of?-catenin in mouse mammary gland causes multiple aggressive mammary carcinomas[40].In colon and other cancers,mutations in the adenomatous polypo-sis coli(APC)tumor suppressor protein or in?-catenin itself,stabilize?-catenin,enhancing its ability to activate transcription of cyclin D1[41]and to increase proliferation [42,43].In most colorectal cancers,mutations in APC result in an absence or loss of function of the protein,so that the phosphorylation of?-catenin by the GSK-3/axin complex is inhibited and?-catenin is not catabolized[44].A high concentration of cytoplasmic?-catenin results in continued growth of colorectal cancer cells[45].In addition,Wnt-2 is up-regulated in gastrointestinal cancer and detection of the Wnt-2protein in the feces may be used as a marker for cancer[46].Mutations in the?-catenin gene and overex-pression of?-catenin are found in most hepatoblastomas [47],and in about50%of human hepatocellular carcinomas (HCC)[27,48,49],as well as in HCC induced by chemicals in mice[50,51].Expression of?-catenin is associated with increased levels of cyclin D1,jun,and myc[51].

It is possible that treatment of cancers with increased ?-catenin as a molecular lesion may be accomplished by agents which cause a decrease in?-catenin.Exisulind and other inhibitors of cyclic GMP phosphodiesterases(sulin-dac sul?de,CP248,CP461)stimulate phosphorylation and degradation of?-catenin,both by proteosome degradation (protein kinase G mediated)and by capase activation,which is independent of GSK-3?,leading to apoptosis of cancer cells[52,53].Since the activities of exisulind and its ana-logues are not only independent of APC and GSK3?,but also of cyclooxygenase expression,p53mutations or bcl-2 expression[54],they may provide effective reversal of the molecular defects of the wnt-signalling pathway in cancers. In early clinical trials,exisulind has tolerable toxicity and shows some promise in combination with other drugs for chemotherapy of metastatic breast cancer[55].In the fu-ture other approaches for therapeutic intervention may be directed to the?-catenin pathway or to other cell signaling pathways.

1.4.Potential/plasticity of stem cells

The ability of a stem or progenitor cell to produce progeny that can express various mature phenotypes is called poten-tial or plasticity.The term totipotent should be reserved for those stem cells that can give rise to all of the differentiated tissues of the body as well as the placenta and membranes (embryonic stem cells;germinal stem cells).The terms mul-tipotent and pluripotent are essentially synonymous:both refer to the ability of a given stem cell to form many differ-ent cell types.In an of?cial NIH primer released in2000, pluripotent is de?ned as“capable of giving rise to most tissues of an organism”,and totipotent as“having unlimited capacity”(https://www.wendangku.net/doc/9f15720943.html,/news/stemcell/primer.htm).During the process of development,the totipotent embryonal stem cells give rise to multi/pluripotent progenitor cells of the germ layers.These,in turn,give rise to oligopotent pro-genitor cells of the developing organs(See Table1).The inner cell-mass cells of the embryo are multi/pluripotent [56].The bone marrow(hematopoietic)stem cells are also multi/pluripotent,and can normally produce red blood cells,platelets,polymorphonuclear leukocytes,monocytes and lymphocyte precursors[57].Most,but not all,of these become terminally differentiated in their fully ma-ture forms.The stromal cells of the bone marrow are also multi/pluripotent and can produce progeny that become

6

S.Sell /Critical Reviews in Oncology/Hematology 51(2004)1–28

Table 1

Terminology of potential (plasticity)Pre?x Meaning Example

Toti-All

Embryonal

Pluri-Several/many Inner cell mass/hematopoietic Multi-Many/much Inner cell mass/hematopoietic Pleo-a Excessive/more Hematopoietic,skin Oligo-Few/little GI stem cell Quadri-Four GI stem cell Tri-Three Bronchial lining Bi-Two Bile duct Uni-One

Prostate

Source:The Random House Dictionary of the English Language.

a Recent evidence indicates that some cells in the tissues used as examples of determined potential may have more potential than previously appreciated (pleopotential).For example,hematopoietic stem cells may give rise to many different mature cell types;skin stem cells,believed to be uni-or bi-potent,may also contain multipotent progenitor cells [63],and thus exhibit pleopotency.

osteoblasts,osteoclasts,chrondrocytes,?broblasts,or mus-cle cells.The intestinal progenitor is quadripotent;it can give rise to progeny that become mucous,absorptive,neu-roendocrine,or Paneth cells.The bronchial lining cells are tripotent:progeny become neuroendocrine,mucous or cili-ated cells.The bile ductular progenitor cells of the liver are bipotent;they produce duct cells and hepatocytes.

Under pathologic conditions,some of these progenitor cells appear to be able to “transdifferentiate”to a differ-ent cell type.For example,bronchial epithelial progenitor cells can produce progeny that become squamous epithe-lium.Hematopoietic and/or bone marrow stromal stem cells may be able to produce progeny that can differentiate into various different tissues including epithelial cells [57].Be-EMBRYONAL

STEM CELL

Fig.4.Relationship between stage of differentiation of cell lineage and cancer type.A hypothetical cell lineage,from totipotent stem cell to terminally differentiated cell,is depicted.It is hypothesized that a malignant event may be manifested at any point along the differentiation pathway.The degree of differentiation of the resultant cancer will be determined by the stage in the process of determination at which the malignant event occurs or is manifested.The concept of tissue stem cells giving rise to cancer represents a modern day version of the “embryonal rest”theory of cancer.Speci?c examples of this model for teratocarcinomas,skin and liver cancer,and hematogenous malignancies are presented in the text.Modi?ed form Pierce et al.[73].

cause the potential of these stem cells is less than totipotent,but more than the expected multi/pluripotency of the hep-atopoietic lineage,the term “pleopotent ”is suggested (see below).

1.5.Adult stem cells and normal tissue renewal

Normal tissue renewal is accomplished by tissue stem cells that divide to give rise to one daughter cell that re-mains a stem cell and another daughter cell that begins the process of determination (asynchronous division).In most organs,the normal replacement of terminally differentiated cells is accomplished by proliferation of progenitor cells or transit-amplifying cells.Transit-amplifying cells provide an expanded population of proliferating tissue determined cells and produce progeny that differentiate into more mature cells that can no longer proliferate and eventually die (see Fig.4).In classic embryology,determination is considered a unidirectional pathway [7].For example,when primitive endoderm becomes committed to forming liver,it is not able to dedifferentiate or to transdifferentiate into another tissue type.Classically,the term transdifferention was used for the process of reprogramming cells that have already embarked on a differentiation pathway,and has been used interchange-ably with metaplasia and transdetermination [58].However,it is now appreciated that adult progenitor cells of one tis-sue may be able to differentiate into mature cells of another tissue type.For example,the adult bone marrow contains small numbers of undifferentiated stem cells having suf?-cient plasticity to give rise to progeny that can become at least nine different mature cell types [19],depending on the environment in which they are seeded [57,59],a concept originally proposed by Cohnheim in 1867[3].According to

S.Sell/Critical Reviews in Oncology/Hematology51(2004)1–287

1. TISSUE DETERMINED ADULT STEM CELLS

2. PLEO/PLURIPOTENT ADULT STEM CELL

3. TRANSDIFFERENTIATON

4. DEDIFFERENTATION

5. FUSION

Fig.5.Five possible pathways for generation of different tissue types in adult tissues.1.From tissue determined stem cells.In the classic model of tissue determination,progenitor cells become committed(determined)for a speci?c tissue type.They then give rise to progeny that differentiate into the speci?c tissue.2.Pleo/pluripotent tissue stem cells.In tissues such as the blood,the tissue stem cell retains the potential to give rise to progeny that can differentiate into different cell lineages.3.Transdifferentiation of determined cells.Progenitor cells that have become determined for one tissue type change their determination and give rise to a different cell type.4.Dedifferentiation.Mature tissue cells,which still have the capacity to divide,undergo backwards differentiation and give rise to progeny that can differentiate into a different cell type.This has been demonstrated during amphibian limb regeneration[314].5.Fusion.It is possible that stem cells from one tissue(e.g.,bone marrow)may fuse with cells of another tissue(liver)to form “hybrid”cells that may re?ect new differentiation properties[164,165].

the original meaning of the term,this is not transdifferentia-

tion,rather,it is unexpected plasticity or“pleopotency”(see

below).Of the possible pathways for differentiation of adult

tissues shown in Fig.5,the most likely paths are1and2,

but not3,4,or5.However,the term transdifferentiation is

frequently used,or,in fact misued,when pleopotency would

be more appropriate.

1.6.Pleopotency of bone marrow stem cells

In1867,Cohnheim hypothesized that the blood contained

cells that can participate in normal tissue renewal of other

organs[3].In fact,it appears from accumulating evidence

that,not only does the bone marrow contain a blood-forming

stem cell,but also it contains an even more primitive cell

with greater potential.This more primitive stem cell pro-

duces not only cells of the hematopoietic lineage,but also

mesenchymal progenitor cells that can give rise to many

other cell types,such as osteocytes,adipocytes,muscle cells,

astrocytes,and neurons,as well as stromal cells that sup-

port hematopoiesis[57,59,60,61].In addition,it has the ca-

pacity to circulate to other organs and to replace different

non-hematopoietic tissues[57,59].Because this cell most

likely is not totipotent like embryonal stem cells,but never-

theless has unexpected potential,the term pleopotent(from

the Greek,excessive,more)is hereby suggested to describe

this cell.Ii is this cell in the adult that offers new approaches

to replacement of damaged tissue not previously considered

possible[62].

The true tissue stem cell normally divides very rarely,but

it can be stimulated to proliferate if there is a loss of,or

an increasing demand on,transit amplifying cells.This is

termed a reserve stem cell.For example,proliferation of the

resting bone marrow stem cells may be activated by blood

loss or by a decrease in the oxygen supply at high altitudes.

To maintain the total number of cells in an adult organ in

equilibrium,the number of progenitor cells that divide must

essentially equal the number of cells that differentiate and

die.If more cells are produced than die,either because the

number of proliferating cells is greater than the number of

cells dying,or because the number of dying cells is smaller

than the number of proliferating cells,then the number of

cells will increase,the primary feature of cancer.Under con-

ditions of temporary stress,such as high altitude oxygen re-

duction or blood loss,this increase is reversible;in cancer

it is not.

2.Stem cells and cancer

2.1.Embryonal rest hypothesis

The histologic resemblance of the tissue of teratocarci-

nomas to that of the developing fetus suggested to Virchow

in the mid-1800s[2,64]a possible relationship between the

two tissues.When compared to normal tissue,cancers were

termed dedifferentiated to denote the less-differentiated cel-

lular structure similar to fetal tissue.Today,pathologists

still refer to the“degree of dedifferentiation”,in the grading

of tumors:poorly-differentiated suggests a bad prognosis;

well-differentiated,a better prognosis.However,it is un-

fortunate that this term implies a process whereby tumors

8S.Sell/Critical Reviews in Oncology/Hematology51(2004)1–28

arise from mature cells becoming immature cells-an unlikely event.Cohnheim[3,4,65]and Durante[5]postulated that cancer in adults develops from embryonal rudiments that are produced in excess and that remain in the tissues of the fully mature organs.In1867,Cohnheim[3]hypothesized that all tissues are renewed by cells in the blood stream;from this concept it follows that both normal tissues and cancer can arise from stem cells in the blood.Beard extended the embryonal rest hypothesis by concluding that tumors arise from displaced placental tissue or activated germinal cells in adult tissues(see Oberling[66]).

In1889,Cohnheim[65]further hypothesized that the be-nign or malignant character of a new growth depends solely on the behavior of the remainder of the organism.He stressed the importance of a blood supply,or angiogenesis,for pro-gression of cells from benign to malignant.Ribbert[67]in 1904proposed that the critical factor for expression of the malignant phenotype of these cells is their isolation from a normal controlling environment,a concept that will be ex-panded in consideration of teratocarcinomas below.Rotter [68]thought that primitive sex cells(germinal cells)wander through the tissue of the developing embryo and can acci-dentally lodge in tissues outside the ultimate sex glands,and can in these new locations serve as the origin of tumors. According to these concepts,all cancers might arise from embryonal-like progenitor cells or germinal cells present in the wrong places in adult tissues.Malignant cells and nor-mal stem cells share the ability to invade,migrate and grow in tissue sites distant from where they arose[69,70].We pos-tulate that tissue stem cells are the modern day equivalent of embryonal rests and that most tumors arise from matura-tion arrest of a cellular lineage derived from a tissue stem cell[6].

2.2.Anaplasia

The term“anaplasia”(ana,backward;plasis,to mold) was?rst coined to describe the change in cells that allows cancerous growth by dedifferentiation.The embryonal rest origin of cancer was generally replaced by the idea that external agents such as chemicals or viruses act on differen-tiated tissue cells to cause cancer,by inducing dedifferenti-ation of mature adult cells.Anaplasia is now used as a mor-phological term to describe a loss of differentiated features (without form),rather than as a term to describe a process. In modern cancer biology,genetic changes leading to alter-ations of growth regulatory functions are widely accepted as causing the altered growth seen in cancers[71].Chemical carcinogens and oncogenic viruses that cause these genetic changes must act on dividing progenitor cells,rather than on mature cells.An exception is the liver,an organ in which even the most mature cells retain the capacity to divide(see below).Thus,even if a carcinogen acts on a mature liver cell,this cell must proliferate in order for the carcinogenic event to be manifested.From a mathematical analysis of the total number of mutation events during the history of a cell,it is evident that mutations are likely to accumulate rapidly in cells during their symmetric linear growth phase (i.e.,during early development[72].It is hypothesized that this process results in the seeding of the tissues of a young individual with a small fraction of mutated stem cells that serve as the origin of cancer in the mature adult.

2.3.Maturation arrest

Cancers at any age may arise from the proliferating progeny of the stem cells(transit amplifying cells);the de-gree of differentiation depends on the stage of differentiation at which the oncogenic lesion is manifested(Fig.4;[6,73]). If maturation arrest occurs early in the determination of a cell lineage,the cancers will be poorly differentiated;if the arrest occurs later,they will be well differentiated.

In order for an alteration in a cell to cause cancer the change must arise in a cell that has the potential to divide and not be lost during normal tissue turnover.For example, the cells above the basal layer of the skin or in the upper mucosa of the gastrointestinal tract that undergo a malignant mutation are most likely sloughed off before they can give rise to a tumor mass.In order for a cancer to be produced, there must be a way for the transformed cells to remain in the body.This is the property of the tissue stem cells or the least-differentiated tissue determined stem cell,which can generate one daughter cell that remains in the tissue.

3.Childhood tumors

3.1.Wilms tumor

Tumors arising in children provide clues to the relation-ship of“residual”embryonic cells and cancer[2].In1899, Wilms[74]described a tumor of the kidney of children un-der8years of age,which contained a mixture of embry-onal cell types.He concluded that it arose from a fragment of the primitive undifferentiated mesodermal tissue,which normally is the anlage of the myotome(striated muscle), nephrotome(Wolf?an body),and smooth muscle.It is com-posed of a mixture of undifferentiated spindle cells,imma-ture epithelial tubules,and“rosettes”of cells similar to em-bryonal glomeruli(James Homer Wright rosettes),as well as sarcomatous tumor cells and nonstriated muscle,leading to the term“nephroblastoma”[75,76].Wilms’tumors ap-pear to arise from embryonal cells in infants;these embry-onic rests differentiate with age and essentially disappear, so that they are no longer a source of cancers after7years of age[76,77].

3.2.Neuroblastomas

Neuroblastomas are tumors of infants composed of a mix-ture undifferentiated spindle cells and immature epithelial tubules[78].Neuroblastomas arise in fetal neural crest cells

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of the sympathetic nervous system.In young infants,they are anaplastic and aggressive,whereas in older children they appear as more differentiated tumors or even as benign ganglioneuromas[79].The undifferentiated embryonal-type neural tumor cells become more differentiated with age,and can differentiate into benign mature cells in the environment of the more mature child.The most likely instance of a tumor arising from stem cells is germinal cell teratocarcinoma. 4.Germinal stem cells and teratocarcinoma

4.1.Totipotent embryonal cancer stem cells

Tumors of germinal stem cells in the adult clearly demonstrate the totipotentiality and tumorigenicity of tu-mor stem cells.Teratocarcinomas of mice can be produced by transplantation of germinal cells from the genital ridges of21-day-old fetal mice into the testes of adult syngeneic mice[80,81],and have been maintained for hundreds of generations by serial transplantation or cultivation in vitro. Through a change in the tissue environment,the normal ger-minal stem cells acquire malignant growth potential.Gard-ner concluded that extrinsic factors in the environment are required to maintain normal differentiation of the germinal cells[58]–a concept avidly espoused earlier by Cohnheim, [65].When removed from this controlling environment,the otherwise normal germinal cells do not mature normally.

4.2.Teratocarcinomas

Teratocarcinomas are made up of many differentiated cell types representing essentially any normal cell of adult tis-sues including germinal cells as well as embryonic and fetal (yolk sac and placental)tissues[82].The malignant cells are the embryonic(embryonal carcinoma)cells[83–85], which form structures resembling pre-somite embryos,so called embryoid bodies(les boutons embryonnaires;[86]). The malignant core cells of teratocarcinomas cultured in vitro also form organized structures that resemble the early developing embryo.The cells that make up the embryoid body are undifferentiated,but have the capacity to differen-tiate into mature benign cells[87,88].Approximately10% of single embryonal carcinoma cells from the teratocarci-noma develop into tumors that can contain more than two dozen well-differentiated adult tissues,including brain,mus-cle,bone,teeth,bone marrow,eyes,secretory glands,skin and intestine,arising from all three germ layers as well as placenta and yolk sac[85].

In humans,teratocarcinomas appear mainly in young adults at any site along the migration pathway of germinal cells from the brain to the gonads during development[82]. The differentiation of the malignant undifferentiated embry-onal carcinoma cells results in an appearance of the tumor that is a caricature of normal tissue development[87,88]. The observation that cancers morphologically resemble the tissues from which they derive was emphasized by Cohn-heim[65](p.751)who wrote:“It is then self-evident that the elements of a tumor cell will closely follow the speci?c type of individual in whose body it arises,for example, in the tumours of man and mammals hairs and not feath-ers will be met with,and in the corresponding tumours of birds,always feathers.”However,the character of the cell of origin of teratocarcinomas,and not the tissue site of origin,determines the cellular makeup of the tumor,but the environment determines the malignant phenotype.The com-position of the mature cells in teratocarcinomas does not differ substantially at the different sites,with the possible exception that malignant ovarian teratocarcinomas appear to contain large amounts of neural tissue[89].Thus,it seems likely that teratocarcinomas arise from an undifferentiated germinal stem cell present in various tissues,usually along the developmental migration pathway of germinal cells. 4.3.Control of malignant potential of embryonal

cancer cells

The growth of teratocarcinomas appears to be due to loss of the growth restrictions that are placed on the developing germinal cells by their normal environment.The ability of the environment to control differentiation of the cancerous cells of teratocarcinomas was unequivocally demonstrated when transplantable teratocarcinoma cells were injected into the inner cell mass of a normal blastocyst[56,90–92].Not only is the blastocyst able to control the differentiation of these tumor cells,but the tumor stem cells are also incorpo-rated into the developing embryo,resulting in development of mosaic adult mice,with normally functioning tissues de-rived from the tumor cells[91].As determined by histocom-patibility antigen type,red blood cell type,immunoglobulin allotypes,and isoenzymes,the cells derived from the tu-mor cells can carry on their normal differentiated functions in the chimera.Remarkably,a male tumor mosaic mouse, named Terry Tom,was able to serve as the father of nor-mal offspring that had the genotype of the tumor cells[91]. This is a common property of normal embryonal stem cells, but has been reported only once for teratocarcinoma stem cells.In this case,the transplantable tumor may be consid-ered to be the grandparent of these F1mice.Regardless of the ability of embryonical carcinoma cells to become func-tioning gametes,the differentiation potential of totipotential cells–and in fact their ability to produce cancers–is con-trolled by environmental signals provided by the appropriate normal tissues,as predicted by Cohnheim[65]and Ribbert

[67].Cancer can be viewed as:blocked ontogeny[93,94],

a problem of the control of the progenitor cell by its niche [13,73],or a matter of chemical morphogens altering stem cell differentiation[95].

Transplantable teratocarcinomas are different from other tumor types in that they have no demonstrable chromosomal abnormalities[56]–an impressive fact,in that it suggests that malignancy in not necessarily an inherent property of

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a cancer cell.The growth and differentiation characteristics of a cancer are also determined by the relationship of the cancer cell to its environment.The control of the growth of some cancers thus depends on speci?c recognition markers between the cancer cells and the surrounding cells.

4.4.Embryonic cells compared to embryonal carcinoma cells

Embryonic stem cells cultured from the inner cell mass of the early embryo[56,96]are similar in many ways to em-bryonal carcinoma(EC)cells.However,they rarely give rise to tumors,they retain a normal karyotype in vitro,and they routinely retain the potential to contribute to the germ line of chimeras,giving rise to gametes that can transmit the embry-onic cell genotype to the next generation[97,98],a property only once reported for EC cells[56,91,99].Although most of the time the malignant potential of the teratocarcinoma cells in the chimeric mice is controlled by the normal en-vironment,some of these chimeric mice do develop tumors [100].This is not found when embryonic stem cells from the inner cell mass are injected into blastocysts[101].Thus there are differences between the embryonal carcinoma cells derived from germinal stem cells and the embryonic stem cells derived from normal embryos and normal germinal stem cells[99].EC cells may acquire karyotypic abnormal-ities when cultured in vitro,but they are unable to transmit speci?c-induced genetic mutations to chimeric mice[102].

4.5.Regulation of tumor cell differentiation

Is the capacity to induce differentiation of malignant cells a general property of cancer cells,or only of teratocarci-noma tumor stem cells?Injection of leukemia cells into the placenta of a10-day-old mouse fetus induced hematopoietic maturation and the appearance of normal white blood cells carrying the leukemia cell marker in the blood of the mature animal[103].Cells of neuroblastoma[104]and melanoma [105]are similarly regulated if placed in embryonal?elds where normal differentiation of these cell types occurs.Thus, it appears that some tumor cell types in addition to terato-carcinomas can be regulated by the appropriate embryonic ?elds appropriate for a given type of tumor.

Normally during development,both cell proliferation and apoptosis are ongoing as the cells of the embryo interact to form the differentiating organs.Could it be that the process that controls the development of normal cells can also be ap-plied to tumor cells?Using giant blastocysts to obtain blas-tocyst?uid[106],it was found that apopotosis of developing cells is caused by the catabolism of polyamines mediated by enzymes such as amine oxidases.The mechanism of apop-tosis is related to the generation of hydrogen peroxide and a developmentally regulated glutathione-dependent protec-tion mechanism[107],which is active not only in the blas-tocyst,but also in the embryonic limb[108].More recently, the hypothesis that embryonic products extracted from the pregnant uterus or from embryos could affect the growth and differentiation of cancers[109,110]has been tested.In vitro, tumor cell lines(glioblastoma,melanoma,renal adenocar-cinoma,breast cancer and lymphoblastic leukemia treated with extracts from zebra?sh embryos obtained during the differentiation stage induced slowing of proliferation,but not when the extract was taken from a mainly replicative stage [111].The application of this interesting approach remains to be worked out.However,the possibility that immature cancer cells can be induced to differentiate has potential for tumor therapy(differentiation therapy;see below).

5.Skin cancer

5.1.Skin cell lineage and malignant phenotype

The cellular type of a skin cancer depends on the stage of differentiation of the epidermal lineage when the cell under-goes malignant change and maturation arrest(Fig.6,for re-cent reviews see[112,113]).The lineage of the skin includes the precursor cells in the bulge of the hair follicle,more determined basal cells in the epidermis,suprabasal prolif-erating cells(transit-amplifying cells)and non-proliferating cells above the transit amplifying cells.Although this is an oversimpli?cation,transformation of the primitive skin progenitor cells in the bulge of the hair follicle gives rise to trichoepitheliomas,which vary in cellular differentiation but usually contain both keratotic and basal regions,as well as clear cells characteristic of hair follicle[114].Ac-tivation of the more determined basal cells of the skin by overexpression of Ras produces squamous cell carcinoma [115,116],and expression of the c-myc gene in normally non-proliferating suprabasal cells reactivates the cell cycle and leads to hyperplasia(papillomas);these,however,do not progress to invasive tumors[117].Different factors appear to be responsible for growth,e.g.Ras,p53,whereas other factors,e.g.?-cantenin for differentiation[113].Mutation in one gene may not be suf?cient for the malignant pheno-type,but increased proliferation associated with a mutation in a growth regulating gene,associated with a muatation in a differentiation controlling gene,may be suf?cient[112]. In squamous cell carcinoma of the uterine cervix associated with human papilloma virus infection,the initially infected and transformed cell is the basal stem cell,but productive infection requires keratinocyte differentiation[118,119]. 5.2.Skin cancers arise in self-renewing stem cells (initiation and promotion)

Perhaps the best evidence that tumors arise from stem or progenitor cells is provided by the two-step model of skin carcinogenesis[120].The two steps are initiation and promotion[121–124].In the classic model,benz(o)pyrene, the initiator,is painted onto the skin.This chemical binds to DNA in the skin cells,causing a permanent genetic al-

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11

HAIR FOLLICULE BULGE CELLS

TERMINALLY

DIFFERENTIATED KERATINIZED CELL

PAPILLOMAS

Fig.6.Skin cell lineage and cancer type.The phenotype of epidermal carcinomas is related to the stage of differentiation of the cell types in the skin where the malignant phenotype is expressed.The most primitive cell is in the bulb of the hair follicle,and the most differentiated cell is the terminally differentiated keratinized cell.

teration (initiation).However,cancers will not arise unless a proliferative stimulus is also given (promotion).This is provided by treating the skin with phorbol ester.Thus,the initiation event induces genetic damage,and the promoter then stimulates the damaged cells to proliferate,leading to cancer.Initiation must occur before promotion.If pro-motion is performed prior to initiation,cancers will not develop.

In the original experiments of Peyton Rous [120,121],initiation was accomplished by painting the ear of a rabbit with coal tar.This produces genetic lesions in the epidermal cells by inducing DNA/carcinogen adducts that cannot be repaired.If no further insult occurs,tumors do not develop.However,if the site is wounded by scraping with a cork borer,epithelial cancer appears at the edge of the wound.The time between initiation and promotion is the critical factor in implicating the stem cell as the initiated cell.This interval can be days,or even months or years in length [122–125].In order for tumors to grow in this model,the initiated cells must survive from the time of initiation to the time of pro-motion.Given the well-established fact that all cells in the skin,except for the self-renewing progenitor cells,turn over completely every 2–3weeks in mice and about 2months in humans [126,127],it is clear that the only way in which the initiated cells could still be present,if months or years have passed since initiation,would be for initiation to have oc-curred in the self-renewing progenitor cell population.In the course of the year or more between initiation and promotion,all of the transit-amplifying cells would have been replaced by newly generated cells from the basal stem cells.Thus,in the initiation-promotion model for skin carcinogenesis,the initiated cell must be a self-renewing progenitor cell.

If this is true,then why don’t the initiated cells grow out and produce cancer during normal tissue renewal?Why is promotion required for initiated cells to grow into cancers?

The answers to these questions are not known.Presumably,normal tissue renewal does not involve proliferation of the long-term self-renewing cells,and any malignant mutations in the transit-amplifying cells will not be expressed as can-cer,since these cells will terminally differentiate.The ini-tiated cell that gives rise to cancer must be a resting stem cell that is only called upon to proliferate under the stress of promotion.

5.3.Field cancerization

The human counterpart of the experimental animal model of initiation and promotion,and the stem cell origin of ep-ithelial cancer,are supported by the phenomenon of ?eld cancerization (Fig.7)[128].Field cancerization was ?rst recognized in humans by the presence of biologically ab-normal tissue surrounding oral squamous cell carcinoma,and the development of multiple primary tumors and locally recurrent cancer in these areas.Field cancerization is asso-ciated with the acquisition of a genetic change in a stem cell,with formation of an altered patch of epithelium which can be recognized histologically by changes such as hyper-keratosis,focal acantholytic dyskeratosis,and acantholysis;these changes re?ect abnormalities of proliferation and/or differentiation of the epithelial cells [129].Patches of ?eld cancerization may have a zone of cells surrounding various epithelial cancers containing mutations of TP53,or showing loss of heterozygosity [130].Thus,?eld cancerization may be considered a manifestation of an initiation event.If fol-lowed by expansion of the initiated population (promotion),a population of cells at high risk for developing further ge-netic changes,leading to invasive cancer is produced.When such a situation is recognized clinically,it is advisable not only to remove any obvious tumors,but also to remove the surrounding “cancerized”?eld,if possible [130].

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CANCER e.g. c-myc

Fig.7.Field Cancerization.Epidermal carcinomas are frequently found to be surrounded by a“?eld”of morphologically altered cells.These cells are believed be changed by mutation or loss of a gene such as p53,which produces abnormalities in proliferation.It is postulated that a second mutation, such as in c-myc,then leads to malignant transformation.

6.Liver cancer

6.1.Participation of various cells in the hepatocyte lineage in hepatocarcinogenesis

Cells at various stages of the liver cell lineage proliferate in response to liver cell injury,or in response to hepato-carcinogens[131,132].The?nding that surgical removal of up to two-thirds of the liver was compensated in a few days by proliferation of hepatocytes,with no evidence of involvement of progenitor cells,demonstrates that mature adult liver cells can proliferate[133–135].On the other hand,carcinogenic studies during the1930s and1940s demonstrated that both formation of nodules of hepatocytes (preneoplastic nodules)and hyperplasia of small round cells in the portal zone of the liver preceded hepatocellu-lar carcinoma(HCC)induced by butter yellow[136–139]. The small cells became known as oval cells[140],but most attention was focused on the preneoplastic nodules as the putative precursor to cancer[140–142],suggest-ing that HCC developed from hepatocytes and not from ductal precursor cells.This concept was supported by the conclusion that bile duct cells and hepatocytes were sepa-rate cell lineages[143].The kinetics of the early elevation of the fetal protein,?-fetoprotein(AFP)during chemical hepatocarcinogenesis gave the?rst clue that various cell populations are involved[131,144,145].When the kinetics of serum AFP elevations was followed during the cyclic N-2-acetylamino?uorene(AAF)feeding model of Teebor and Becker[142],AFP became elevated before nodules appeared[146].Localization of the AFP-positive cells and proliferating cells indicated that periductular cells were the ?rst cells to proliferate and to express AFP during carcino-genesis in studies using a choline de?cient diet[147–150]. However,different carcinogenic regimens induced pro-liferation of non-ductular and ductular oval cells preced-ing HCCs[151,152].Then,using a modi?cation of the Solt-Farber[153]model,a number of investigators demon-strated proliferation of oval cells predominately in ducts [151,154–158].A review of the various cellular changes preceding HCC in experimental hepatocarcinogenesis in rats has led to the conclusion that different cells in the liver lineage,including mature hepatocytes,biliary progenitor cells,and periductular cells,can give rise to HCC(Fig.8). The presence of periductular primitive liver stem cells [159],and their early proliferation during hepatocarcinogen-esis[148,149],has stimulated an interest in pleopotenital bone marrow cells as to the possible origin of these cells [132].

HEMATOPOIETIC

STEM CELL

?

Fig.8.Postulated stages of the hepatocytic lineage that may respond to liver injury or carcinogenic protocols.Following various models of liver injury or chemical hepatocarcinogenesis different cell types in the hep-atocytic lineage may respond:1Undifferentiated periductular oval cells, which may arise from circulating bone marrow precursor cells.2.Periduc-tular cells intrinsic to the liver,3.Bipolar ductal progenitor cells,or 4.Mature hepatocytes,which retain the potential to divide.Periductular cells respond to periportal injury induced by allyl alcohol or to choline de?ciency-ethionine carcinogenesis.Bipolar ductal progenitor cells re-spond to injury and to carcinogenic regimens,such as the Solt-Farber model,when proliferation of hepatocytes is inhibited.Hepatocytes re-spond to partial hepatectomy and to carcinogensis by diethylnitrosamine (DEN)(from[132,315]).

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Given the?nding that hepatocytes can arise from bone marrow precursors[160–163],it seems likely that circu-lating bone marrow-derived stem cells that enter the liver will do so in the periductular region,via the hepatic artery or the portal vein.Is it possible that these circulating bone marrow-derived cells may not only give rise to cells in the hepatocytic lineage,but also to HCC[132]?

6.2.Fusion of bone marrow and liver cells

The possibility that bone marrow stem cells might fuse with recipient liver cells in vivo was raised when it was re-ported that sex mismatched syngeneic bone marrow cells fused with recipient liver cells during restoration of chroni-cally damaged liver in fumarylactoacetate hydrolase(FAH) knockout mice(Tyrosinemia)[164,165].In these recipient mice,there is remarkable ongoing chronic liver damage and proliferation with polyploidy of recipient cells,raising some doubts about the origin of the“fused cells”.There are con-?licting reports on the signi?cance of fusion in normal re-placement of liver cells.Both Newsone et al.[166]and Ishikawa et al.[167]found that human cord blood mononu-clear cells could give rise to human hepatocytes in recipi-ent immune de?cient NOD/SCID mice,with no evidence of fusion.For example,Newsone et al.[166]examined5620 HepPar1(human maker)positive cells in the NOD/SCID mouse liver;each of these cells had nuclear staining char-acteristic of human nuclei and contained only human DNA by FISH analysis.In more sophisticated experiments us-ing cre-lox donor recipient models,both positive and nega-tive fusion results have been obtained.Alarez-Dolando et al. [168]counted both non-fused donor cells and fused cells, but Ianus et al.[169]?nd no evidence of fusion.If fusion does occur,and hepatocellular cancers arise from bone mar-row stem cells that have fused with hepatocytes,this could explain the marked variations in ploidy seen in HCC.

7.Adenocarcinomas

7.1.Stem cell origin

The observations on the stem cell origin of teratocarci-nomas and HCCs may be extrapolated to other organs[6]. Exocrine glands consist of acini of glandular cells drained by a collecting duct.The key progenitor cells are located between the gland and the duct.For example,it is known, both from histologic examination of human lesions[170]and from examination of“preneoplastic”lesions of experimental mouse models[171,172],that breast cancers arise from an undifferentiated suprabasal progenitor cells above the my-oepithelial cells in the duct or the terminal ductular lobular unit.The cells in the terminal lobular unit are the same cells that proliferate during pregnancy and produce progeny that eventually differentiate to form the lactating mammary gland [172].Prostate cancer also arises from suprabasal stem cells [173–175].Intestinal carcinogenesis is directed toward func-tionally anchored stem cells in the crypts[176],and mutated stem cells appear to be able to spread through the mucosa, by fusion of crypts,before becoming invasive[177].Muta-tion in the adenomatous polyposis coli gene(APC)is asso-ciated with increased expression of survivin and?-catenin, which allows expansion of crypt stem cells and development of colon cancer[178].Finally,mixed tumors of epithelial and mesenchymal components(carcinosarcomas)appear to arise from a common monoclonal origin[179].In each in-stance,carcinomas arise from tissue progenitor cells.Is it possible that these cells arise from circulating stem cells that have the ability to cross basement membranes,and to local-ize in various glandular tissues?One of the best examples of the role of maturation arrest of tissue progenitor cells (transit-amplifying cells),due to malignant mutations in the primitive stem cell is that of the hematopoietic system. 8.Hematopoietic stem cells and leukemia

8.1.Normal blood renewal

Malignant cancers of the blood provide one of the clearest examples of the role of stem cells in cancer.Blood is one of the most rapidly replaced tissues in the body[180].In1917, Pappenhein[181]postulated the existence of an undifferen-tiated stem cell for blood cells,“gemeinsame Stammzelle”. These hematopoietic or blood-forming stem cells are located in the bone marrow.The ability of the bone marrow progen-itor cells to form colonies of various types of blood cells in the spleen after transplantation was?rst recognized by Till and McCullough[182]in1961.The frequency of these pro-genitor cells could be estimated from the number of colony forming units[183].By the use of such techniques,it was possible to show that a very small number of progenitor cells can both reproduce and give rise to the complete spectrum of phenotypic colonies of blood cells[184].The number of proliferating hematopoietic progenitor cells has been esti-mated to be0.05%of the total number of bone marrow cells [185];the number of non-proliferating parental stem cells for the proliferating cells is even less.

8.2.Multi/pluripotent hematopoietic stem cells

The multi/pluripotent most primitive hematopoietic cell in the bone marrow is a resting stem cell,which normally does not proliferate,but which can respond to stress,such as the loss of blood or adjustment to a low oxygen environ-ment(high altitude).The primitive hematopoietic stem cell is the parental cell for the major population of proliferating cells in the bone marrow;the transit-amplifying progeni-tor cells(Fig.9;[186]).The progenitor cells,in turn,give rise to progeny that differentiate to mature circulating blood cells[19].Circulating blood cells vary from polymorphon-clear cells,which only live for a day or two,to erythrocytes,

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T R A N S I T A M P L I F I C A T I O N

PLEOPOTENT LONG-TERM STEM CELL (very rare) (CD34-, c-Kit+, Sca1+)

Fig.9.Hematopoietic Cell Lineage.Suggested differentiation pathway of bone marrow stem cells in the hematopoietic pathway.The multi/pluripotent stem cell is a reserve stem cell and normally proliferates very rarely.The expansion of the cell lineage is accomplished by transit amplifying cells that expand into nine different cell lineages.The number of multi/pluripotent stem cells may be very low,whereas the number of various differentiated cell types is very high.

which survive for months,to lymphocytes,which survive for days to years [187].The majority of circulating blood cells cannot proliferate.For example,erythrocytes have excluded their nuclei,and polymophonulclear cells have inactivated their nuclei and condensed them into clumps.Because of the large number of polymorphonuclear cells and their rapid turnover,a very large number of precursor cells is required to generate polymorphonuclear cells.These are present in the bone marrow as transit-amplifying blast cells existing at various stages of differentiation into mature polymorphonu-clear cells.A close relative of the polymorphonuclear cells,the circulating monocytes,are derived from a common pro-genitor cell,but retain the ability to proliferate.8.3.Bone marrow transplantation and pleotypic potential

After bone marrow transplantation,the donor bone mar-row cells can give rise to circulating cells that have the potential to differentiate into unexpected cell lineages (pleopotential),including:endothelium [188–191],muscle [192],liver [157,160–168,193],pancreatic islet beta cells [169,194,195],heart [196,197],brain [198–202],type I [203]and type II pneumocytes [162],kidney parenchyma [204],skin [63,205],ocular retina [206],glomerular en-dothelial cells [207,208],glomerular mesangial cells [209],and other organs [162].Although the bone-marrow cells from which these various cell types have been derived have markers of the hematpoietic stem cell [162,163],it has not been ruled out that this pleopotent cell is of stromal origin [210–212].Serial transplantation indicates that a sin-gle bone marrow cell may give rise to many different tissue

types [57,162]and suggests that a common precursor must exist,not only for the stromal and hematopoietic lineages,but also for other germ layer-derived cell types [187].It is this putative pleopotent bone marrow cell that has stimu-lated the great revival of interest in adult stem cells in the last few years.Although it is dif?cult to prove that there are not different stem cells for different tissues in the bone marrow,at least three studies demonstrates that single bone marrow stem cells can give rise to different epithelial tissue types after serial transplantation [57,162,191].Regardless if there is a single pleopotent cells or multiple stem cells with different potential,the putative stem cells arising from the bone marrow of adults may have just as much promise as embryonic stem cells for stem cell therapy [213–215].8.4.Potentiality caveats

There are some caveats to the generally accepted assump-tion of pleo/multi/pluripotentiality of tissue stem cells,in-cluding hematopoietic stem cells.For example,although it was reported that transplanted bone-marrow stem cells pro-vided new endothelium in transplant models of atheroscle-rosis,more careful analysis in one model suggests that the new endothelial cells were actually derived from pre-existing endothelium [216].In a study of muscle-derived cells con-tributing to bone marrow,the muscle-derived bone marrow stem cells were found to be derived from hematopoietic stem cells present in the muscle [217].Whereas Bjornson et al.[218]generated substantial numbers of hematopoietic pre-cursor cells from neural stem cells,Morshead et al.[219]could not reproduce this result (see Nature Med.8(3002)535–537).In addition,recent studies have shown that in

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vitro culture of adult mouse brain or bone marrow stem cells with embryonic cells generates hybrid cells containing chro-mosomes of both adult and embryonic cells[220,221],thus raising the question of whether adult stem cells make other tissues by fusing with existing tissue cells.Both of these studies employed co-culture of adult cells with embryonic cells,which is not the case in bone marrow transplants in adults.However,this?nding has stimulated in vivo stud-ies investigating the possibility that transplanted donor bone marrow cells fuse with recipient cells in various organs.As mentioned above,the results are con?icting.In a chronic model of restoration of damaged liver,fusion of bone mar-row cells and liver cells was described[164,165],whereas fusion was not seen in restored pancreatic islet cells[169], in hepatocyes of immune-de?cient(NOD-SCID)mice re-ceiving human bone marrow cells[166,167],or in the buc-cal mucosa of female recipients of male bone marrow cells [222].The demonstration of fusion in the chronic severe in-jury models may be due to marked disruption of the archi-tecture of the liver and compression of the circulating cells against damage cells.Thus,the possibility of fusion in vivo, in the absence of extensive cell injury or in mature diploid cells[57],remains to be resolved(see[223]).

8.5.Unanswered questions

Assuming that pleopotent bone marrow stem cells actu-ally exist,a number of basic questions remain unanswered. Is there a single cell type in the bone marrow that is pleopo-tent,or are there various types of“progenitor”cells that can give rise to different cell types in other organs?It appears that a single cell that can do it all[57,162,191].Is the abil-ity of a bone marrow stem cell to contribute to an epithe-lial organ a very rare or a more common event?Krause and colleagues[57]?nd it to be a fairly common occurrence; whereas Wagers and colleagues[224]claim that this is an extremely rare event.The ability of bone marrow-derived cells to contribute to epithelial tissue may depend on the age of the donor and the method of puri?cation of the stem cells,but clearly the weight of published evidence in favor of a pleopotent bone marrow stem cell outweighs the nega-tive evidence[225].Does the progenitor cell normally cir-culate,or is it liberated from the bone marrow by humoral signals after injury?The evidence is that this cell circulates normally,but that its numbers may be increased after injury. Do progenitor cells normally exist in mature organs,such as liver or kidney,and can they re-circulate back to the bone marrow?Are these cells derived from the bone marrow,or can totipotent progenitor cells be generated in differentiated organs[226]?The ability of brain cells to become blood cells[218,227]suggests that the answer to the latter ques-tion is“yes”,but re-circulation of blood-derived cells to the brain,and back to the bone marrow,cannot be ruled out. Can progenitor cell lines that could be used for transplanta-tion,for organ replacement,or for delivery of gene therapy, be derived from bone marrow or other organs[187]?Ex-tensive culture of neural stem cells may be required before such cells can differentiate into blood cells,suggesting that rare differentiation events may occur upon culturing;and that these events may account for neural to blood differen-tiation[219].This?nding suggests that normal brain pre-cursor cells cannot contribute to blood-cell lineages in vivo. Answers to these questions,and clinical applications of the use of tissue stem cells,will undoubtedly be forthcoming in the next few years[187,225].

9.Leukemias as models of maturation arrest of a cell lineage

9.1.Leukemia as a stem cell cancer

Leukemias are caused by a genetic change or in a single cell that produces progeny that continue to proliferate,and/or do not die[228].The demonstration that tumor growth de-pends on a subpopulation of proliferating stem cells(cancer stem cells)in the tumor was?rst described in transmissi-ble leukemias of mice.Furth and Kahn[229]in1937were able to transplant leukemia from one mouse to another us-ing a single undifferentiated cell.In1955,Makino and Kano [230]obtained clones of tumor cells from single cells.Hu-man leukemias feature gene rearrangements that are present in all the cells of the tumor[231,232],suggesting the tumor’s origin in a single progenitor cell that has undergone a ma-lignant gene rearrangement.This concept also holds true for leukemias and lymphomas of humans(for a recent review, see[233]).The effect of a genetic change in the precursor cell of the population is exempli?ed by the malignant in-crease of multiple cell types in chronic mono/myelogenous leukemias,including various types of polymorphonuclear cells(neutrophils,eosinophils,and basophils),as well as monoctyes,erythrocytes,and platelets(megakaryocytes),all of which contain the same genetic lesion or lesions.Thus, the malignant cell is the multi/pluripotent tumor stem or pro-genitor cell with the capacity to differentiate into mutiple types of blood cells[234].

9.2.Chemotherapy and reserve stem cells

The pattern of response to chemotherapy of acute leukemias also implies that these cancers arise from a prim-itive stem cell[234].Repeated cycles of anti-proliferative treatment are required to affect resting tumor cells that are not cycling at one speci?c time of therapy.Since the ther-apy acts on actively proliferating cells,one cycle of drug treatment should be suf?cient to eliminate the actively pro-liferating cells present at the time of treatment.However, the malignant genetic change is also present in the“resting”or G0stage stem cells that are not proliferating when the drug is given.Successful treatment may require four or more cycles of anti-proliferative therapy to catch all of the leukemic cells.Even so,up to30%of patients will require

16S.Sell /Critical Reviews in Oncology/Hematology 51(2004)1–28

Table 2

Some gene rearrangements in leukemias and lymphomas Leukemia/lymphoma Gene rearrangement Gene activated

CML t9:22bcr/abl Tyrosine phosphorylase AML t8:21IL-3R

Tyrosine kinase

APL t15:17PML/RAR ?Retinoic acid receptor,blocks diff.ALL

t12:22TEL/MN1Transcription factors t9:12TEL/abl Tyrosine phosphorylase Burkitt’s (B-cell)t8:14IgG/myc c-myc (transcription)B-cell lymphoma t14/18IgG/bcl2bcl-2,blocks apotosis CLL

?

?

blocks apotosis

Listed are only a few of the many gene rearrangements found in leukemias and lymphomas.These rearrangements frequently result in translocation of a promoter and a structural gene,resulting in activation of expression of the rearranged gene.The two major classes are those that activate kinases and cause proliferation,and those that activate genes (such as bcl-2),that block apoptosis.Examples of how these gene rearrangements are manifested in leukemias and lymphomas are presented in Figs.10and 11.

total bone marrow ablation and stem cell transplantation from a normal donor,in order to eliminate all of the primi-tive resting cells that have the genetic lesion.This implies that the leukemia arises in the reserve hematopoietic stem cell,which can only be eliminated by total bone marrow ablation.

9.3.Gene translocations and leukemias

The characteristics of various leukemias illustrate the relationship between the stage of maturation arrest of the cells and the genetic change;this determines the natural history (growth rate)of the tumor.A few of the known

gene

EOSINOPHILS NEUTROPHILS BASOPHILS

, IL-6

IL-5LIF

ERYTHROCYTES

Fig.10.Constitutive activation of the IL-3receptor causes myelogenous leukemia.IL-3is active in the proliferation and differentiation of all cells in the myeloid series,including polymophonuclear leukocytes,monocytes,megakarocytes (platelets),and erythrocytes.Thus,constitutive activation of the IL-3receptor results in an increase in the precursors of all of these cell types,a characteristic of acute myelogenous leukemia.Modi?ed from [316].

rearrangements in leukemias and lymphomas are listed in Table 2.Leukemias feature genetic lesions that affect either cell proliferation (transactivation)or cell death (apoptosis).The nature of the genetic lesion can be directly related to the type of leukemia [228,231,235,236].

9.3.1.The IL-3receptor

In acute myelogenous leukemia (AML)there is often a gene rearrangement leading to constitutive activation of the IL-3receptor resulting in activation of tyrosine kinase and cell proliferation.The role of IL-3in normal hematopoiesis is shown in Fig.10.From this ?gure it can be appreci-ated that activation of the IL-3receptor should produce

S.Sell/Critical Reviews in Oncology/Hematology51(2004)1–2817

Fig.11.Activation of immunoglobulin promoter linked to oncogene(c-Myc)and/or apoptosis blocking gene(Bcl-2)produces B-cell cancer in transgenic mouse model of Burkitt’s lymphoma.B-cell lymphomas occur when the immunoglobulin gene promoter is linked to an oncogene(c-Myc)and/or a gene that codes for the apoptosis blocker,Bcl-2.Although the Ig-promoter/oncogene translocation is present in every cell in the animal,the promoter is only activated in B-cells,so that other cells in the lymphocytic lineage are not affected.

an increase in all cells in the myeloid lineage,the charac-teristic of AML.With respect to determining the stage of differentiation at which the genetic rearrangement is found, the rearrangement is generally identi?able in the early myelogenous progenitor or even in the more primitive bone marrow stem cells(see above).

9.3.2.B-cell lymphomas and the immunoglobulin heavy chain gene promoter

The role of translocation of the immunoglobulin(Ig)gene promoter with either activating or apoptosis-blocking genes for B-cell lymphoma is illustrated in Fig.11.In transgenic mice,translocation of the Ig-gene promoter to the c-Myc and/or Bcl-2gene may produce a B-cell lymphoma[237] similar to Burkitt’s lymphoma[238].In this model it is pre-sumed that the genetic lesion is present in all cells.However, the effect of the gene is not manifested until the Ig-heavy chain gene promoter is activated in B-cells.Thus,activation of the oncogene c-Myc or the apoptosis blocker Bcl-2linked to the Ig promoter does not occur until the Ig-gene promoter to which they are linked is activated.When this occurs,there is either increased proliferation(c-Myc)or decreased apop-tosis(Bcl-2),or both,resulting in a rapid increase in the number of immature B-cells that do not die.Thus,the ge-netic lesion causing leukemias occurs in the stem cells,but the growth characteristics of the tumor are determined by the stage of maturation of the B-cell lineage in which the Ig-heavy chain gene promoter is activated.

9.4.Stage of maturation arrest and clinical course of leukemias

The degree of differentiation and the clinical behavior of a cancer are directly related to the stage of maturation arrest of the cells in the tumor lineage(Fig.4).The clinical course of a given leukemia depends on the rate of cell proliferation (growth fraction)and the rate of cell death.A high percent-age of large blast cells is a characteristic of fast-growing acute leukemias,with little or no terminal differentiation, whereas a high percentage of mature differentiated cells is a characteristic of chronic leukemia(Fig.12).Blast cells represent cells that are in the cell cycle;mature cells repre-sent cells that are not in the cycle.A leukemia composed of blast cells has a high growth fraction(a high percentage of cells in cycle at any given time).A leukemia composed of mature cells,as in chronic lymphocytic leukemia,has a low growth fraction.A mixture of blast and more mature cells implies an intermediate growth fraction.In chronic lympho-cytic leukemia(CLL)most of the cells are resting G o lym-phocytes,with fewer than1in1000cells in cycle at any time,but the cells of CLL do not die,and instead accumu-late over years.

The clinical course of leukemia is determined by the rate of replacement of the normal bone marrow progenitor cells with leukemic progenitor cells.This replacement results in a loss of the normal cells and their function.Loss of red blood cells results in anemia;loss of platelets leads to bleeding; loss of polymorphonuclear cells causes decreased resistance to infection.Leukemia with a high growth fraction will re-place the normal progenitor rapidly(in months),whereas leukemia with a low growth fraction may take years to pro-duce symptoms(Fig.12).Thus,the histologic classi?cation and the clinical course of leukemia are determined by the stage of arrest of differentiation where the genetic lesion is activated.Depending on when the lesion was acquired,it may be presumed that the genetic lesion is present in many other cells of the body,including the pleopotent bone mar-row stem cell,but the malignant phenotype is only expressed when the genetic lesion is activated.If it is activated early in the course of maturation,most of the cells will re?ect the properties of actively dividing transit-amplifying cells, such as in the multi/pluripotent hematopoietic progenitor

18S.Sell /Critical Reviews in Oncology/Hematology 51(2004)

1–28

Fig.12.Growth fractions,morphology,and clinical course of three selected leukemias.In acute myelogenous leukemia,the tumor cells are arrested in an active growth phase:cells that divide and do not enter G 0,but pass directly into the next cell cycle.Few,if any,differentiated cells are seen;the tumor is made up of blast cells.The growth fraction is very high,the expansion of cells is essentially exponential,and the time to death,if the disease is not treated,is within a few months.In chronic myelogenous leukemia,the arrest is at the level of the transit-amplifying cells.The number of cycling cells is much smaller,and a much higher proportion of the tumor cells undergoes differentiation so that many cells at various stages of differentiation are seem.The growth fraction is small,and the time to death is years.In chronic lymphocytic leukemia,maturation arrest occurs in small non-dividing cells.The functional change is a lack of cell death.The vast majority of the cells are in G 0,so that the growth fraction is vanishing small,and the time to death is decades.Modi?ed from [317].

cells of acute myelogenous leukemia.If the genetic lesion is manifested at a more mature stage of maturation,a chronic leukemia such as CLL will result.

10.Differentiation therapy

If the malignant cells of cancers are cancer stem cells [6,180,233,239–241],then it should be possible to treat can-cers by inducing differentiation of the stem cells,i.e.,dif-ferentiation therapy [242].As discussed above,the tumor stem cells of teratocarcinomas can be in?uenced by the en-vironment of the developing embryo to differentiate into normal adult tissues.If tumor cells can be forced to dif-ferentiate and to cease proliferation,then their malignant potential will be controlled.Although a number of agents have been studied over the years [243],the most thoroughly examined and clinically tested as a differentiating agent is retinoic acid (RA,Vitamin A),in particular all-trans-retinoic acid (ATRA).

10.1.Retinoic acids and teratocarcinomas

Ironically,although teratocarcinomas were the ?rst tu-mors that were shown to differentiate in vitro after addition of low concentrations of RA [244],this has not proven to be an effective treatment in vivo.Addition of retinoic acid (RA)or RA in combination with dibutyryl-cAMP,stimulates dif-ferentiation of a cell line of nondifferentiatied,immortalized embryonal carcinoma cells,called F9,to an epithelial cell type consistent with extra-embryonic endoderm [244–246].RA induces differentiation through activation of speci?c nuclear receptors,RAR,which activate c-Fos/c-Jun medi-ated transcription [247,248],resulting in upregulation of the expression of differentiation genes [249–251](Fig.13),including AFP [252].At least three nuclear retinoic acid

S.Sell/Critical Reviews in Oncology/Hematology51(2004)1–2819

Signaling/

Induces Senescence

Inhibits Proliferation

Fig.13.Retinoic acid(RA)reacts with nuclear receptors in embryonal carcinoma cells leading to inhibition of proliferation and induction of proliferation.RA and related compounds pass into the nucleus where they react with nuclear receptors(RAR-RXR)that from a transcription complex with c-Fos,c-Jun,AP-1,and most likely other activation proteins resulting it upregularion of a number of genes related to inhibition of proliferation and induction of differentiation.

receptors(RAR)-?,-?,and-?,and three retinoid X re-ceptors(RXR)-?,-?,-?have been identi?ed.The most active receptor appears to be a heterodimer between RAR and RXR[253].The RAR-RXR heterodimer binds to DNA sequences called retinoic acid response elements found in the promoter region of target genes and enhance gene transcription after binding their ligands[254,255].A num-ber of cellular pathways are affected during RA-induced activation of RAR.A speci?c candidate tumor suppressor gene product,Disabled-2,is induced after RA treatment. Disabled-2greatly decreases c-Fos expression and disas-sociates MAPK activation,resulting in differentiation of the cells[256].RA-induced differentiation correlates with accumulation of the cyclin-kinase inhibitors p21/Waf1and p27/Kip[257],a decrease in cyclin D3[258],and activation of the?-catenin/Lef-Tcf signaling pathway[259].In addi-tion,RA-induced differentiation of human teratocarcinoma cells leads to increased methylation of the hTERT promoter and silencing of the hTERT gene,which codes for the catalytic subunit of telomerase[260].Differentiation of F9 cells is also induced by depleting putrescine and spermidine, required for polyamine synthesis[261].Thus,RA activates multiple pathways leading to decreased proliferation and increased differentiation of embryonal carcinoma cells. However,treatment of teratocarcinomas that demonstrate sensitivity of cells in vitro to retinoids,has not yielded sat-isfactory results in vivo[262].Some cells may differentiate, but the malignant core of cells remains and quickly grows back.The treatment of choice for human teratocarcinoma is surgery and,if total resection is not possible,follow up treatment with irradiation and/or cisplatin-based cytotoxic chemotherapy[263].The ef?cacy of the treatment is re-lated to the initial tumor burden.Almost80%of patients with germ cell tumors treated with cisplatin remain relapse free for2years,but successful treatment of recurrent dis-ease also depends on tumor burden[264,265].Combination chemotherapy that includes other drugs,such as taxol,may have an effect on refractory cases[265,266].Treatment of refractory testicular cancer with trans-retinoic acid has not proved to be effective even in combination with cisplatin [267].

10.2.Retinoids and acute promyelocytic leukemia(APL) APL is a cancer of children wherein the precursor cells of the myelocytic lineage,expressing markers of the M3type, fail to mature,accumulate in the bone marrow and blood in enormous numbers,and eventually force out production of normal blood cells.The molecular lesion in APL is a t(15:17)gene rearrangement involving the nuclear retinoid acid receptor alpha(RAR?).In this rearrangement the gene for a protein called promyelocytic leukemia protein(PML) is fused to the RAR?gene(PML-RAR?).PML is normally found in a discrete,circumscribed nuclear structure called a nuclear body in cells of the myeloid series[268,269].The PML-RAR?fusion product inhibits the RAR?[270],dis-rupts the nuclear bodies,and[271],and without its ligand, retinoic acid,the fusion product functions as a constitutive transcriptional repressor,blocking hematopoietic differen-tiation and allowing the accumulation of poorly differen-tiated leukemia cells[272].On the other hand,the APL fusion gene product appears to induce genes that maintain the stem cell phenotype and represses DNA repair genes, resulting in a mutator phenotype that enhances tumor pro-gression[273].ATRA treatment upregulates the ubiquitin activating enzyme-E1like protein(UBE1L)that triggers degradation of the PML/RAR?fusion protein[274],acti-vates RAR?mediated transcription,allows reformation of the PML-nuclear body and stimulates differentiation and eventual apoptosis of APL cells[271,275].

Currently about90%of newly diagnosed patients with APL achieve complete remission and over70%are cured by ATRA therapy[276]with or without concomitant chemotherapy with methotrexate and cytarabine[277].The clinical presentation of APL is frequently associated with hemorrhage and low platelet counts due to the decreased ability of the bone marrow to produce platelets.Poor prog-nostic factors include older age,elevated white blood cell count,low platelets,and CD56expression[278].The overall survival rate is greatly increased by ATRA versus chemotherapy alone and there is an up to75%disease free ?ve year survival rate for both induction and maintenance on ATRA treatment[279].After achievement of complete remission,maintenance therapy with ATRA with low-dose chemotherapy is may be useful.Despite overall great suc-cess in treating APL with retinoids,relapse with develop-ment of acquired resistance to retinoid induced maturation is not uncommon and is responsible for treatment failure [280,281].The mechanism of resistance to RA therapy is not well understood.It may be due to increased ATRA metabolism,increased expression of the RA binding pro-teins,P-gylcoprotein expression,or mutations in the ligand binding domain of RAR-?[282].Due to severe side ef-fects of ATRA treatment(see below)dosing schedules are

20S.Sell/Critical Reviews in Oncology/Hematology51(2004)1–28

critical to the success of treatment.Treatment with other differentiating-inducing agents,cytotoxic or chromatin re-modeling agents,as well as receptor-selective and modi?ed retinoids may overcome this resistance[280].Cytotoxic treatment with arsenic trioxide is the treatment of choice for refractory ATRA resistance[277,278].In addition,re-sistance can be overcome to some extent by combining histone deacetylase inhibitors,such as sodium butyrate, with RA.This treatment facilitates RA-induced gene tran-scription and induction of apoptosis,but has little effect on granulocytic differentiation of the promyelocytic leukemia cells[283].Bone marrow transplantation may be important in the future for patients in relapsed/refractory disease or those in a second CR[278].

10.3.Retinoid differentiation therapy;other cancers

In addition to embryonic cells,teratocarcinomas,and leukemias,RA induces differentiation in other cell types [278,284–288].In vitro,retinoids reduce the ability of human melanoma cells to form colonies[289]and this inhibition correlated in a small number of patients with a modest clinical response[290].Transfection of RAR into non-responsive cells in vitro can restore responsiveness to retinoids by antagonizing activator protein-1(AP-1), blocking proliferation and activating differentiation[291]. Retinoids may be able to upregulate expression of p53 and induce p53dependent G1arrest and apoptosis of human lung cancer cells[292].In solid cancers RA in-creases differentiation and apoptosis,and decreases pro-liferation(blockade of G1),invasiveness,and metastasis [274],Retinoids have been employed with limited success in treatment of many different solid cancers[287],includ-ing squamous cell carcinoma[293],adenocarcinoma of the prostate[294,295],neuroblastomas[296],HCC[297]in humans,and breast carcinoma in mice[298].Treatment of squamous tumor cell lines with ATRA may act though the RAR to upregulate a putative class II tumor suppres-sor gene,tazarotene-induced gene3(TIG3)[299].13-cis RA may be used in conjunction with other agents,such as inferferon-?,?-tocopherol and cisplatin,for treatment of advanced squamous cell carcinoma[300,301].After ini-tial clinical remission,solid tumors,like APLs,frequently develop resistance to retinoid-induced maturation[282]. 10.4.Chemoprevention of cancer by retinoids

If retinoids can induce differentiation of malignant tumor stem cells,then it is even more likely that they could in-duce differentiation in so-called“premalignant”cells and be used as chemopreventive agents for cancer[287].In addition to the premalignant changes seen in the bronchial epithe-lium of smokers,RA has been used with some success to cause regression of laryngeal papillomatosis and oral leuko-plakia[302].Loss of nuclear retinoid acid receptor(RAR)in bronchial epithelium is considered a marker for preneoplasia frequently found in smokers and an indicator of increased risk for lung cancer[303].Many premalignant and malig-nant cells have a reduced expression of RAR?,and treatment with retinoids may induce RAR?and induce differentiation in some cancer cell lines[292]and in biopsy specimens of bronchial epithelium of smokers[304].Treatment with 9-cis-RA can restore RAR expression in former smokers, raising the possibility that this could be a chemopreventive agent for lung cancer[305].In an earlier study,treatment of squamous cell cancers and preneoplastic lesions with retinoids had only a modest clinical effect,and these studies were limited by dose-related toxic effects,including dermati-tis,emotional liability and headache[306].More sophisti-cated studies using different retinoids and other inhibitors, such as growth factors receptor,are proposed([307],but a statistically signi?cant chemopreventive effect has not yet been demonstrated.

10.5.Retinoic acid syndrome

Finally,it seems that there is a price to pay for any approach to cancer therapy.Many patients treated with all-trans retinoic acid develop“retinoic acid syndrome”[308].This syndrome consists of fever,weight gain,respi-ratory distress,interstitial pulmonary in?ltrates,pleural and pericardial effusion,episodic hypotension,and acute renal failure.Recently the recommended dosage of ATRA used for treatment of APL has been reduced from45to25mg/m2 day in an attempt to limit these side effects[309].When identi?ed early,this syndrome can be effectively treated with dexamethasone[288].

11.Summary and conclusions

It is the hypothesis of this paper that cancers arise from stem cells in adult tissues.All adult tissues are made up of lineages of cells consisting of tissue-determined stem cells and their progeny(transit-amplifying cells),that provide cellular lineages producing many cells that differentiate to the mature functioning cells of the tissue.Each tissue has an individual rate of normal tissue renewal.Tissues with a rapid turnover of cells,such as blood and gastrointestinal lining epithelium,have a relatively large number of imma-ture transit amplifying cells.Other tissues,such as the liver or brain,with a very low normal renewal rate,have very few transit-amplifying cells.In any case,all tissues have stem cells,but these stem cells do not all have identical pos-sibilities(potentials)for producing different cell types.For example,the bone marrow may contain a stem cell that can unexpectedly give rise to various tissue types including ep-ithelial and neural tissues(pleopotent),as well as a stem cell that give rise to blood cells(multi/pluripotentent).The gas-trointestinal lining contains stem cells that can give rise to at least four different cell types(oligopotent),whereas the liver stem cells appear to give rise to two cell types(bipotent).

干细胞存储行业研究报告精编版

2017年干细胞存储行业研究报告 因为具有潜在的再生能力，干细胞自其诞生之日起，就被认为可能是革命性的医疗技术。2007年和2012年的诺贝尔生理或医学奖，分别授予了与干细胞有关的发现。 2011年开始，干细胞概念再次被市场热炒。根据Market Research及Transparency Market Research预计，到2018年全球干细胞储存市场容量将增长至181.6亿美元。 干细胞存储业务的想象空间虽然不及干细胞治疗，但依然是现阶段许多干细胞公司的重要业务。对投资机构来说，一方面存储业务充沛的现金流可以缓解研发费用的压力，一方面细胞分离等技术能力的高低也能侧面反映公司的技术水平，对于分析标的的风险和潜力意义重大，值得持续关注和研究。 一、概述 干细胞是具有自我更新、高度增殖、多项分化潜能及良好组织相容性等特点的细胞群体。因为具有潜在的再生能力，干细胞自其诞生之日起，就被认为可能是革命性的医疗技术。2007年和2012年的诺贝尔生理或医学奖，分别授予了“在利用胚胎干细胞引入特异性基因修饰的原理上的发现”和“发现成熟细胞可被重写成多功能细胞，细胞核重编程技术（iPS细胞）”两项与干细胞有关的发现。 2011年开始，干细胞概念再次被市场热炒。在A股市场上，以中源协和为代表的企业当年股价飞涨。 存储业务涉及的主要是造血干细胞和间充质干细胞，前者主要来源于骨髓、外周血、脐带血、胎盘等，后者主要来源于骨髓、脐带、脂肪、脐带血、羊膜、胎盘等，进而形成了脐带血造血干细胞等细分种类。目前干细胞存储的主要市场仍在新生儿相关的业务，随着分离等技术的成熟和临床研究的深入，新生儿干细胞存储种类正在从脐带血造血干细胞转向胎盘、脐带等来源的间充质干细胞。

win7系统动态桌面设置全攻略

Win7系统动态桌面设置全攻略 您是否厌倦了不会动的电脑桌面？即使是Win7系统自带的自动切换壁纸功能，时间长了也觉得没什么新意，无非就是像幻灯片一样切换而已。实际上，Windows 7 还有一个更棒的隐藏功能——可以将任何WMV格式的视频设置为电脑桌面并循环播放，而且没有时间长度的限制！ 试想，当你的同事、家人或朋友路过你的电脑时，发现你的桌面竟然会动！而且呈现的是唯美细腻的高清视频！他们的脸上会是什么表情？从今以后，他们对你的敬仰，一定会犹如滔滔江水，连绵不绝...... 打开这一隐藏功能的方法其实很简单，只需对着屏幕喊：“芝麻开门！桌面动起来！”您的图片壁纸就变成更为生动的视频壁纸了。。。呵呵，开个玩笑，后面我们会告诉您正确方法。当然，Windows 7 系统也确实有语音识别功能，而且挺有意思的，有兴趣的朋友可以点击阅读《Win7真好玩：超有趣的"语音识别"功能》一文。 点击图片查看下一页

动态GIF图，图片打开速度较慢，请稍等 在开始介绍设置方法之前，先请您看看动态桌面的最终效果，然后您再决定是否继续看下去： 视频：动态桌面的实际效果 怎么样，感觉如何？如果您也觉得这个功能很炫，愿意尝试一下的话，请看下面的图文教程，其实非常简单，正常情况下只需两分钟就可以搞定了。

动态桌面补丁的下载和设置过程 言归正传，打开这一隐藏功能只需一个小补丁，它叫做“Windows7-DreamScene”，我们去百度上搜这个名字或是“动态桌面”都能找到，它的体积很小，只有221KB而已。 如果您是保存到桌面上的话，下载完毕后桌面上就会多出一个名为 Windows7-DreamScene.RAR的压缩文件，我们将其解压后，就可以看到这个小补丁的本体了。双击它，就会打开下面这个窗口： 看到这个窗口后，随便按键盘上的一个键即可，比如回车键按任意键后，这个小窗口的内容会不断发生变化，我们不用管它，大概20秒后变化完全停止，我们再按任意键，这个窗口就会消失，暗示我们该补丁已安装成功，Win7的隐藏功能已被打开。

干细胞与组织工程

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