Chromosomal Proteins in Transformed and Neoplastia Cells: a Review1

[CANCER RESEARCH 38, 1181-1201, May 1978] Chromosomal Proteins in Transformed and Neoplastia Cells: A Review1

Gary S. Stein, Janet L. Stein, and Judith A. Thomson Departments of Biochemistry and Molecular Biology Â¡G.S. S., J. A. T.] and Immunology and Medical Microbiology Â¡J.L. SJ, University of Florida, Gainesville, Florida 32610

Studies carried out in the 1960's by Huang ef al. (186) and It is becoming increasingly apparent that chromosomal proteins (histones and NHCP2) play an important role in by Allfrey ef al. (8) provided the first biochemical evidence dictating structural and functional properties of the eukar- that histones are represser macromolecules. Huang and yotic genome. The histones, or basic chromosomal pro Bonner showed that, when increasing amounts of histones teins, have been shown to function as repressors of DMA- are complexed with DNA, a progressive decrease in DNA- dependent RNA synthesis as well as to be involved in dependent RNA synthesis is observed and that transcription packaging of the genome. Components of the NHCP also is completely inhibited at a 1:1 histone:DNA ratio. Allfrey ef may be involved in the maintenance of genome structure, al. (8) demonstrated that progressive removal of histones but additionally several lines of evidence suggest that from nuclei results in an activation of RNA transcription. among this complex and heterogeneous class of chromo While numerous lines of evidence have substantiated a somal proteins are macromolecules that are responsible for repressor function for histones, their limited heterogeneity rendering defined genetic sequences transcribable. The makes it unlikely that histones can, by themselves, recog structural and functional properties of chromosomal pro nize specific gene loci. The similarity of histones in all teins have been the subject of several reviews (27, 99, 110, tissues of an organism is a clear indication of the lack of 231, 397, 414, 415) and monographs (56, 63, 180, 410, 417) specificity of these proteins. Although in a few instances an and will therefore be discussed here only briefly. additional histone has been identified (e.g., H5 in avian Understanding the manner in which chromosomal pro erythrocytes) and in several lower eukaryotes specific his teins interact with the information encoded in the nucleo- tone fractions appear to be absent, these situations consti fide sequences of the DMA double helix undoubtedly will tute isolated exceptions. The presence of only 2 substitu enhance our comprehension of a broad spectrum of normal tions in the entire amino acid sequence of 1 arginine-rich biological processes, such as growth, development, differ histone fraction (H4) that has been compared in calf thymus entiation, and maintenance of cellular phenotype. Further and in pea bud (89) additionally suggests the evolutionary more, it is reasonable to anticipate that elucidation of the stability of the histones and the structural and functional mechanisms by which gene readout is controlled will facili constraints that have been imposed on mutational variabil tate deciphering of the basis for the aberrations in gene ity of these molecules. expression that accompany disease processes such as Some heterogeneity exists in individual histone fractions; neoplasia. The primary emphasis of this review will be the this is largely attributable to modifications of the proteins in potential involvement of chromosomal proteins in the onset the nucleus or in the cytoplasm of cells following comple and maintenance of the neoplastic state. tion of synthesis. Without changes in the amino acid se quences of histones, acetate (summarized in Ref. 348), Histones phosphate (16,165, 244, 270), and methyl groups (6, 42, 60, 61, 312) may be covalently added to and removed from the Five defined chromosomal proteins have historically been amino acids of certain histones. Additionally, the sulfur in designated as histones. The histones are metabolically cysteine can be modified. In the case of phosphorylation, stable, positively charged chromosomal proteins enriched while it has been known for some time that phosphate in arginine and lysine residues and completely lacking in groups may be posttranslationally added to serine and the amino acid tryptophan. Histones as a total class are threonine, it has been observed recently that acid-labile associated with DNA in approximately a 1:1 ratio (w/w) and amino phosphate is associated with other amino acids (71, are intimately involved in structural and functional proper 387). ties of the genome. The 5 principal classes of histones are, Posttranslational modifications of histone fractions in with few exceptions, present throughout the plant and cells have been correlated with changes in transcriptional, animal kingdoms. Although all 5 histones bear a net positive replicative, and structural properties of the genome [re charge, sequence analysis reveals that each histone ex viewed by Hnilica (180)]. These modifications in the his hibits a different nonrandom distribution of amino acids. tones are thought to alter histone-DNA binding and histone- This asymmetrical arrangement of amino acids in the var histone relationships as well as the modes of interaction of ious histones provides an important clue to the mode of histones with other genome-associated proteins. Acetyla- interaction of specific histones with one another and with tion and phosphorylation of histones precede or occur other genome components. concomitantly with the activation of transcription associ 1Studies from our laboratory described in this review were supported by ated with numerous biological processes, e.g., stimulation Research Grants CA-18874 from the NIH, BMS-7518583 from the National of cellular proliferation, stimulation by hormones, develop Science Foundation, and F75UF-4 from the American Cancer Society. 2The abbreviations used are: NHCP, nonhistone chromosomal proteins; ment, and transformation by oncogenic viruses. Changes cyclic AMP, cyclic adenosine 3':5'-monophosphate; EBV, Epstein-Barr virus. in the phosphorylation of specific histones also occur at Received April 7, 1976; accepted January 10, 1978. defined points during the cell cycle (165). Removal of

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1978 American Association for Cancer Research. G. S. Stein et al. acÃ©tateand phosphate groups from histories which occurs genome, the 2 properties often being extremely difficult to as a function of time may provide a mechanism for repres separate. sion of activated genetic sequences. Recent studies have From the brief preceding discussion, it should be evident provided evidence that the metabolism of phosphate and that histones play an important role in the maintenance of acetate groups associated with defined amino acid residues genome structure and in the repression of DNA-dependent of histone fractions is regulated by specific enzymes (238, RNA synthesis. When histone-DNA interactions are altered, 245). modifications in gene readout and in the structural organi Attention recently has been focused on the arrangement zation of the genome are observed. These findings lead to of histones along the DNA molecule and their role in the conclusion that histones are structural as well as regu chromatin structure. Electron microscopic and biochemical latory macromolecules. However, their limited heterogene evidence from several laboratories has revealed clusters ity and lack of specificity suggest that histones do not by of protein approximately 100 A in diameter containing 2 themselves possess the ability to recognize defined gene each of histones H2A, H2B, H3, and H4 and 1 or possibly 2 loci. molecules of H1 histone (110, 145, 179, 230, 232, 255, 294, 303, 311, 325, 342, 391, 466). These clusters, referred to as "v bodies" or "nucleosomes," are associated with DNA Nonhistone Chromosomal Proteins approximately 200 base pairs long, and it appears that the Recently, considerable attention has been focused on the DNA is located on the external aspect of the particles. Such NHCP portion of the genome due to evidence suggesting histone:DNA complexes have been observed in all plant and that among these complex and heterogeneous proteins are animal cells thus far examined and have also been reported macromolecules that are involved in the regulation of gene in some DNA tumor viruses (135, 152, 262, 325). Stretches expression. The chemical and biological properties of the of nucleotides that are nuclease sensitive (80, 81) are NHCP have been extensively reviewed (27, 63, 99, 205, 267, presumably located between the clusters. When examined 317, 319, 397, 404, 410, 414, 416, 419). under the electron microscope, the observed configuration Amino acid analysis of the NHCP as a total class reveals is that of beads on a string with spaces, presumably DNA, an enrichment in acidic amino acid residues, principally between the nucleosome particles. However, the packing glutamic acid and aspartic acid. However, the individual ratio of DNA and protein cross-linking data suggest that components of this class consist of acidic, basic, and adjacent nucleosomes are contiguous, perhaps joined by neutral proteins. In contrast to the histones, which are H1 histone. This apparent contradiction may be explained extremely stable proteins, the nonhistone chromosomal if one assumes that the nucleosomes are separated during proteins exhibit a spectrum of stabilities with half-lives preparation of nuclei and chromatin for electron micros ranging from several minutes to several cell generations copy. The localization of the NHCP in relation to the (43, 88). Furthermore, unlike the histones, the synthesis of nucleosomes is only beginning to be elucidated. The in which is tightly coupled with DNA replication, various volvement of histones in chromatin structure has recently classes of NHCP are synthesized throughout the cell cycle been reviewed by Kornberg (231). (36, 43, 212, 324, 352, 401, 405), and their synthesis is It has been observed that the genetic complexity of the generally unaffected by inhibition of DNA replication (405, DNA (diversity of genetic sequences) associated with iso 418). lated nucleosomes is the same as that of the total DNA Many of the NHCP can undergo posttranslational modifi (242). This finding suggests that the histone complexes are cations including acetylation (424), methylation (125), and located randomly along the DNA and that no specific DNA phosphorylation (225, 226). Chains of polyfadenosine di- sequence is required for nucleosome formation. The lack phosphate ribose) are found associated with certain NHCP of preferential association of histones with defined genetic (47, 67, 389, 422), and glycoproteins (413) as well as glycos- sequences is supported by formation of nucleosome-type aminoglycans (413) have been shown to be components of complexes between histones and viral as well as bacterial the NHCP. Additionally, cysteine-containing chromosomal DNA's. Furthermore, the frequency of sequences coding proteins exhibit oxidation of sulfhydryl groups and reduc for mRNA's is the same in DNA of isolated nucleosomes as tion of disulfide bonds (158). These posttranslational modi it is in the total nuclear DNA, suggesting that template- fications confer the potential for increased specificity on active regions of the DNA are also associated with the the NHCP. However, direct evidence for a functional rela histone complexes (242). Other recent evidence has sug tionship between these posttranslational modifications of gested, however, that there may be some subtle differences chromosomal proteins and alterations in gene expression in the association of chromosomal proteins with actively remains to be established. transcribed sequences. These studies have shown that Functionally, the NHCP can be defined as structural, globin and ovalbumin genes in tissues that are actively enzymatic, and regulatory macromolecules. Genome-asso transcribing these mRNA's have a different sensitivity to ciated contractile proteins (94, 203, 251, 252) may in part nuclease digestion than do globin or ovalbumin genes in assume structural responsibility, although this role is prob tissues that are not synthesizing globin-specific or ovalbu- ably not fulfilled solely by contractile proteins. Modifica min-specific RNA, respectively (15, 131, 465). The relation tions in the circular dichroism spectrum of chromatin fol ships among nucleosomes, transcription, and units of lowing extraction of 0.25 M NaCI-soluble NHCP suggest that replication clearly warrant further study. Probing these components of this class of chromosomal proteins are questions may produce important information regarding involved in determining structural properties of the genome the structural and functional properties of the eukaryotic (77, 256). The principal enzyme systems associated with the

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NHCP are summarized in Table 1; the variety of complex ated by restriction enzyme cleavage and amplified in plas- enzyme systems reflects the functional diversity of these mids, will be used for such fractionation procedures. How proteins. ever, caution should be exercised in assuming that the Quantitative and qualitative differences in NHCP have types of protein-DNA interactions that are observed by been correlated with alterations in the biological states of these affinity methods are those that occur in the nuclei of cells and tissues that reflect modifications in gene expres intact cells. sion. Such differences in the NHCP are consistent with a Phosphorylation of defined molecular weight classes of regulatory role for these macromolecules and have been NHCP has been correlated with modifications in gene extensively discussed in several reviews (27, 99, 267, 319, activity in numerous biological systems (225, 226, 414). 397, 404, 410, 414, 416). Increased amounts of NHCP have These findings suggest that phosphorylation of NHCP may been observed in metabolically and transcriptionally active be an important aspect of the mechanism by which genetic compared with inactive tissues (93). An increased sequences are regulated. Phosphorylation of NHCP is ap NHCP:DNA ratio is also found in transcriptionally active parently catalyzed by both cyclic AMP-dependent and cyclic (euchromatin) compared with inactive (heterochromatin) AMP-independent (222) protein kinases. An important ques chromatin (37, 124, 146, 272, 274, 338). Qualitative varia tion remaining to be resolved that relates to NHCP phos tions in NHCP reflect their species and tissue specificity phorylation and genetic control is whether specificity (98, 266, 341, 374, 433, 459, 469) as well as differences in resides with the particular NHCP that are phosphorylated or active compared with inactive chromatin (146). Addi with the protein kinases that catalyze the phosphorylation tionally, changes in the composition and metabolism of reactions. Recent studies that have shown differences be various molecular weight classes of NHCP accompany tween NHCP kinases of normal and neoplastic tissues modifications in gene expression associated with develop suggest that regulation of NHCP phosphorylation may at ment (204, 374), differentiation both in vivo (323, 451) and in least in part reside with the phosphorylating enzymes (435). vitro (420, 488), stimulation of cell proliferation (254, 344, The quantitative and qualitative variations in NHCP just 403, 406, 407, 441), progression through the cell cycle (36, discussed indeed suggest a regulatory role for these mac 136, 405), response to steroid hormones (171, 201, 276, romolecules. However, the evidence is of a correlative 277, 304, 305, 340, 396, 425, 440) and various pharmaco nature and therefore circumstantial. In vitro transcription logical agents (347), viral infection and transformation (78, studies and analysis of RNA's synthesized under cell-free 234, 235, 256, 345, 412, 487), cellular aging (407), and a conditions have permitted a more direct, but not conclu broad range of other biological phenomena. sive, assessment of the involvement of NHCP in the control The selective affinity of proteins for binding to DNA, of gene readout. presumably by recognizing and interacting with particular Initial attempts at examining the role of NHCP in the nucleotide sequences, has provided further evidence for regulation of transcription were made by assessing the specificity of NHCP (7, 227, 316, 433). DNA affinity methods effects of NHCP on DNA-dependent RNA synthesis. Results provide a powerful method for fractionation and purifica from several laboratories indicated that NHCP, when added tion of chromosomal proteins, and it is anticipated that in directly to histone:DNA complexes or to various chromatin the near future specific genes, isolated from DNA fraction preparations, stimulated the availability of DNA as template

Table 1 Enzyme components of the NHCP Ref. Function Nucleic acids as substrates DNA polymerases 70, 184, 185,261. 315. 442, Polymerization of deoxyribonucleotides into DNA 443 RNA polymerases 86, 130, 250, 284, 428, 467 Polymerization of ribonucleotides into RNA Nucleases 299, 445 Processing or degradation of RNA and/or DNA Nucleotide ligase 133 Joining of DNA segments during DNA replication and repair Nucleotide exotransferases 355, 398, 457 Addition of nucleotides to the ends of nucleic acids Methylases Methylation of DNA or RNA Chromosomal proteins as substrates Proteases 26, 65, 79, 132 Processing or degradation of proteins Methylases 62, 84 Methylation of histones and NHCP Kinases 71,72, 222,387,426 Phosphorylation of histones and NHCP Poly(adenosine 174,422,473 Addition of ADP ribose moieties to chromosomal dÂ¡phosphateribose) proteins polymerase Poly(adenosine 174,283 Removal of ADP ribose moieties from diphosphate ribose) chromosomal proteins glycohydrolase Acetylases 129, 331, 332 Acetylation of histones and NHCP Deacetylases 238, 450 Removal of acetate groups from histones and NHCP

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for transcription of RNA (Refs. 210, 246, 394, 395, and 458; we will discuss alterations in the NHCP that are associated reviewed in RÃ©f.410). While these early results were en with transformation and tumorigenesis. The possible in couraging, they are somewhat inconclusive due to forma volvement of such modifications of chromosomal proteins tion of nonspecific aggregates. Further results that suggest in the alteration of gene expression in neoplasia will be a direct involvement of NHCP in transcriptional control considered. have been provided by chromatin reconstitution experi ments where it appears that the aggregation problem can Gene Regulation and Neoplasia be overcome. The isolated eukaryotic genome (chromatin) can be dissociated and fractionated into its principal com It has been postulated that cancer is a disease of gene ponents (DMA, histones, and NHCP) and then, with a regulation (83, 269, 462). This would imply that the abnor technique developed by Bekhor ef al. (30) and Huang ef al. malities seen in cancer are due to the malfunctioning of the (186), chromatin can be reconstituted with selected genome very complex and undefined mechanisms that dictate the components. Essentially, DMA, histones, and NHCP are gene expression of a given cell at the particular time of its combined in high-salt:urea, and the salt is progressively life cycle. The malfunctioning of these mechanisms would removed by stepwise dialysis, followed by removal of the allow for the derepression or repression of genes in a urea. With the techniques of chromatin reconstitution, in manner that is prohibited in the normal differentiated cell. vitro transcription, and analysis of RNA transcripts by hy Although the causative factors may be quite varied and bridization to [3H]DNA's complementary to defined mRNA's, unrelated, the manifestations of the disease would be solely evidence has been obtained that suggests that a compo due to the disruption of these regulating mechanisms. If nent^) of the NHCP may be involved in the tissue-specific cancer can then be explained by the untimely or abnormal transcription of globin genes (24, 76, 318), the steroid expression of "normal" genes (genes that are expressed in hormone-induced transcription of ovalbumin genes (439, some cell types and contribute to the natural development 440), and the cell cycle stage-specific transcription of his- or sustenance of the organism), then one would expect that tone genes (200, 402). The similarity of native and reconsti all aspects of cancer cells would be exhibited by some cell tuted chromatin preparations is suggested by quantitative at some time during the life cycle of the organism. To and qualitative evaluations of the chromosomal proteins, substantiate this postulate let us briefly examine the major assessment of transcriptional properties, and utilization of characteristics and macromolecules exhibited by neoplas- probes for structural integrity (30, 320, 409, 411). However, tic cells and briefly review the evidence that these proper the choice of tissues or cells and the methods for prepara ties are found in some types of "normal" cells. tion of nuclei and chromatin are extremely important. In The major distinguishable features of cancer cells (inva- some situations utilization of protease inhibitors appears to siveness, metastasis, rapid cell growth, and escape from be essential. the host immune system) are also the properties of many In evaluation of results from transcription studies in embryonic cells. Invasiveness is the prime characteristic of which native and reconstituted chromatins are used, the the trophoblast (220), and tumor cells are similar to troph- following crucial points must be considered. RNA associ oblasts because both invade nondecidualized tissue but not ated with chromatin preparations may provide a back decidual tissue (471). In addition, a specific protease, plas- ground level of RNA sequences that can interfere with minogen activator, which recently has been correlated with quantitation of results from transcription experiments. the invasibility of cells, has been demonstrated in both Chromatin-associated RNA's may also cofractionate with trophoblast and malignant cells (326, 375, 472). During NHCP and thus interfere with interpretation of results from embryogenesis many cells dissociate themselves from sur reconstitution experiments. Additionally, to date most chro rounding cells, migrate through tissues, and establish matin transcription studies have been executed with the themselves in a new position. This is a situation analogous use of bacterial RNA polymerase. It is quite possible that to that of metastasis of tumor cells (83). Alterations accom there is an additional level of regulation that exists in the panying transformation include changes in the cell surface. intact cell and that can be recognized only by the appropri These changes can be shown by the property of neoplastic ate homologous eukaryotic RNA polymerase. While indeed cells to bind preferentially lectins, such as concanavalin A the techniques of in vitro transcription and reconstitution or wheat germ agglutinin, to a much greater extent than do of chromatin may have limitations, with appropriate con normal adult cells (52). After exposure to agglutinins trans trols they provide the most effective system presently avail formed cells have been shown to exhibit a more normal able for evaluating the contribution of genome-associated growth pattern in culture (52). Embryonic cells are also proteins to expression of genetic sequences. agglutinated by concanavalin A and by wheat germ agglu In the preceding discussion we have attempted to sum tinin after proteolytic treatment to uncover the binding sites marize briefly some highlights of progress that has been (286). made during the past several years toward elucidating the Tumors have long been known to produce a factor that role of chromosomal proteins in determining the structural stimulates the host to provide a blood supply (14, 120, 121, and functional properties of the genome. With regard to the 150). This angiogenesis factor is also found in the placenta regulation of gene expression, histones appear to act as (119). A characteristic of some human tumors that can nonspecific repressers of DNA-dependent RNA synthesis. sometimes lead to detection is radiogallium uptake, and In contrast, among the complex and heterogeneous NHCP although the mechanisms of localization are unknown this are components that appear to regulate the transcription of phenomenon has also been observed in embryonic tissues specific genetic sequences. In the remainder of this article, (96, 310). Tumors containing many tumor-specific and fetal

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1978 American Association for Cancer Research. Chromosomal Proteins and Gene Expression antigens can escape destruction by the host immune re The partial sequence of the a-fetoprotein molecule has sponse. Since this is also a characteristic of fetal tissues been determined (350). The relationship of a-fetoprotein to (189, 368, 444), which are known to induce antibodies in cell proliferation (388) is being currently studied, although the mother (209), a common escape mechanism has been there is some controversy as to whether the synthesis and/ suggested for both fetal and neoplastic tissues (83, 209, or levels of a-fetoprotein are coupled to specific periods of 381). the cell cycle. The relationship of a-fetoprotein to hormone After examination of the cancer cell on a molecular level, binding is also being examined (446) and may be especially many macromolecules have been identified in tumors that important since hormones are intimately involved with the are characteristic of other adult cell types or of fetal tissue. control of gene expression at several levels. Transcriptional Included in these macromolecules are cellular isoenzymes capacity of chromatin, specific RNA species, and the NHCP (87, 373, 462). Many fetal isoenzymes have been shown to have all been shown to change after exposure to hormones. be produced by tumors (83, 363), and stomach, brain, and In addition hormones have been shown to have an effect at muscle isoenzymes have been identified in many hepato- the level of translation (372). mas (363). The research on isoenzymes is so extensive that Numerous other oncofetal antigens have been defined it is presented as Table 2 and is necessarily incomplete. (50,53, 57, 140, 148, 168-170, 194,275,289,321,328,436, However, sufficient evidence is presented to substantiate 454). By the use of radioimmunoassay techniques, detec the idea that tumor cells exhibit the resurgence of fetal tion of a ferroprotein (a2H-globulin), normally found in fetal molecular forms of enzymes and the expression of heterol- organs and in fetal serum, has been found in greater than ogous cellular isoenzymes. An example of such an isoen- 80% of children with tumors (50). Another antigen that may zyme that has received attention with regard to its diagnos be of diagnostic value is the carcinoembryonic antigen, tic capabilities is the "Regan" form of alkaline phosphatase found in human fetal gut and in a high percentage of human (69, 113-117, 421). This isoenzyme, distinguishable from colon cancers (97, 137-139, 173, 285, 349, 453). Additional the placental isoenzyme, has been found in patients with examples of embryonic analogs and antigens are presented cancer of various organs, including lung, gastrointestinal in Table 2. Also listed are certain forms of RNA species that tract, and genitals. The frequency of occurrence of the are common to embryonic and tumor tissues. Regan alkaline phosphatase in human cancer seems to be An outstanding example of expression in neoplasia of about 10%. Cultured human cancer cells (HeLa TCRC-1) normally repressed genetic information is the observation also express the carcinoplacental Regan isoenzyme (380), of the decondensation and reactivation of the normally although it has been reported recently that, when this cell condensed X chromosomes in human female cervical and line is grown in immunosuppressed rats, the Regan isoen- gastric carcinomas (434). This may be a relapse to a fetal zyme disappears and is replaced by an isoenzyme resem state since it is known that both X chromosomes must be bling adult bone and liver types (379). Return of the cells to active at some time during embryogenesis for normal de tissue culture results in the return of the Regan isoenzyme velopment of the fetus (264). suggesting that the altered gene expression in malignant Teratocarcinomas represent an excellent system for the states may not be irreversible. study of the correlation between embryonic gene expres Many embryonic and fetal analogs of adult proteins have sion and malignant gene expression in vitro (48, 92, 107, been observed in neoplastic tissues (2, 75), an example 163,199, 260). Cell lines of teratocarcinomas can be derived being the appearance of fetal hemoglobin found in leuke in several ways. Testicular teratomas and ovarian teratomas mia and a few other hematological diseases (279, 377, 378). occur spontaneously in mouse strains 129 and 2T, respec There are also a rapidly increasing number of embryonic tively. Teratomas can also be induced by grafting either and fetal antigens found to be associated with neoplastic early embryos or genital ridges into the testes. Some of tissue (54, 73, 101, 118, 138, 249, 263, 478, 486), although these teratomas can then be serially transplanted in synge- fetal antigens can also be found in some nonneoplastic neic adult mice. Injection of tumor pieces i.p. leads to the conditions (202). The most extensively studied example is formation of so-called embryoid bodies found in the ascites o-fetoprotein produced by hepatocellular carcinomas and fluid. These embryoid bodies contain embryonic-like cells embryonal cell carcinomas of testis and ovaries (1, 2, 138, termed EC cells. EC cells are malignant but can differentiate 371) which was first described by Abelev et al. (3). The into derivatives of all 3 germ layers; they can even be development of techniques for the detection of minute transferred into mouse blastocysts and can participate in amounts of a-fetoprotein, including enzyme immunoassay, the development of the blastocysts into adult mice (48, radioimmunoassay (31, 172, 221), immunoperoxidase (164), 193). These results are quite interesting since they suggest immunofluorescent probes (164, 302, 460), and counter- that the embryonic environment may control the autono immunoelectrophoresis (229), has led to the use of a-feto mous proliferative capacities of the malignant cells. Tera- protein as a diagnostic tool. This tumor-associated antigen tocarcinoma cells, when injected into adult mice, result in can be detected by methods of radioimmunoassay in death in a relatively short time. Hence, the study of terato greater than 70% of patients with hepatocellular carcinoma carcinomas might not only further our understanding of the and greater than 60% of patients with teratocarcinomas interrelationship of embryonic and malignant gene expres (147). Recent studies have been initiated towards ex sion but also help uncover a mechanism for decreasing the amining a-fetoprotein and its expression at the molecular proliferative abilities of malignant cells. Characteristics of level. The isolation of the mRNA for a-fetoprotein has been these cell lines have been investigated by Nicolas ef al. reported, and it appears that there may be polyadenylated (290), and the distribution of certain enzyme activities was as well as nonpolyadenylated forms of the message (427). studied in cloned teratoma-derived cells infected with SV40

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Table 2 Common components to normal and neoplastia cells tissue or cell of ComponentA. originColon originPlacentatissue or cell of Isoenzymes ThymidinekinasePyruvate cancer HeLacellsKB HumancellHuman fetal 206, 399,42922, cellsHuman fibroblasts spleenFetal SV40Humantransformed with rhabdomyosarcoma Wilms'tumorBladder adenocarcinomaHepatomaRat kinaseAlcohol tissueRat 47535364-367,23, 105, 456,

dehydrogenase hepatomasFast-growing stomach (fastmigrating)Aldolase Rat fetalliverRat

CHexokinase hepatomas fetalliverFetal 423217240166, Human rhabdomyosarcomas Yoshida ascites hepatoma ZajdelahepatomaUterine ascites

IIUridine type carcinomasNovikoff tissue, intestine, heart, skeletalmuscleFetal

kinase (low- hepatomaEhrlichascites liverFetal form)Carbamylphosphatemolecular-weight

ascites carcinomas liverType 483300167, 273, IIBranched-chainsynthetase type HepatomasYoshida

amino hepatoma3'-MeDAB"ascites I: fetal liver acid transferases types 1 TypebrainRat III: rat 301191,300,301356, andIIBranched-chain

amino primary tumors brain IIIGlycogenacid transferases type Morris hepatomas Chemical carcinogen-treated Rat ovary rat hepatocytes Spontaneously transformed RatplacentaMuscleMusclePlacentaRef.353 cellsAscites

synthetase hepatomas AH 66F 357358, (muscleform)Phosphorylase and AH130Poorly

(muscle differentiated hepato 359113-116, type)Alkaline masHuman phosphatase (Re bronchiogenic carci 421 gan form)Neoplastic noma Human lung, gastrointestinalNormal 117,278 tract Genital tract tumors Human hepatocellular Carcinoma of stomach Fructose-1,6-diphospha- Rapidly growing hepatomas Muscle of several species 360,361 tase (muscle type) Ehrlich ascites

type)B.Glutaminase (kidney hepatoma 183, 214, 228, 258 Novikoff hepatoma FetalliverFetal LC 18 hepatoma MammarycarcinomasHuman Fetal antigens and analogs a-FetoproteinCarcinoembryonic hepatocellular carci liver1,2,138,263,371Fetus nomaHuman antigenÂ«2H-GlobulinAH-130 gastrointestinal tu 97,137,139,173,285,349,453Fetal morsSerum

of children with tu organ 50 mors (81%)Kidney

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Table 2-Continued

Neoplastic tissue or cell of Component origin Normal tissue or cell of origin Ref. Fetal hemoglobin Leukemia and other hemato- Fetus 279 logical diseases

Intestinal antigen Gastric neoplasia Fetal stomach and intestine 289

Fetal sulfoglycoprotein Gastric cancer Fetus 168-170

Nonspecific fetal antigens Rat: Novikoff hepatoma Fetal tissues 57 Chemically induced mouse 328, 454 sarcomas Mice: 3-methylcholanthrene sarcomas 140 Mice: polyoma tumor 321, 436 Colorectal carcinomas Embryonic tissues 383 Germinal cell tumors of testis Fetal tissues

Membrane antigen Human melanoma Fetal brain tissues 194

Plasminogen activator SV40-transformed rat embryo Mouse embryo 326, 375 cells

/3-Oncofetal antigen Colon carcinoma Fetal organs 126 Breast carcinoma Melanoma Endometrial carcinoma

Placental antigens (PA-1) SV40-transformed rabbit kid Placenta 68 ney cells (TRK-1)

6S DNA polymerase 3'-MeDAB-induced hepato- Rat embryos 75 mas Neonatal liver Morris hepatoma Regenerating liver

Nuclear protein DNA anti 3'-MeDAB-induced hepato- Embryonic tissues 73 gens mas

Isoferritins Malignant tissues Early fetal tissues 12

Tissue polypeptide anti Malignant tissues Placenta 38, 263 gen

C. RNA species tRNA's Mouse plasma cell tumors Fetal liver 21, 41,54,55,241 tRNA methyltransferases Morris hepatomas Undifferentiated stem cell 128, 142, 151, 175 Ehrlich ascites tumor 215,216,343.468,476 Mouse fibrosarcomas Reuber hepatoma cells 3'-MeDAB, /V,/v-dimethyl-p-(m-tolylazo)aniline. to allow for the propagation of the cell line (438). The latter liver (382)] and tryptophan oxygenase (108, 334) genes cell lines (SVTER) may be useful for the study of malignant during hepatocarcinogenesis. Feigelson et al. have shown cells as well as for the study of developmental pathways that the lack of synthesis of Â«2u-globulin during hepatocar (438). cinogenesis is the result of the lack of functional mRNA for Illustrating the property of malignant cells to express this protein. This could be further evidence of altered genetic information normally repressed prior to the onset transcriptional capacities of neoplastic tissues. Another of cancer but not fetal related is the production of hor example of altered gene expression could be the coupled mones by nonendocrine tumors (44, 45, 100, 103, 141, 144, appearance of the mRNA for an RNA tumor virus (the 259, 265, 271, 351, 393, 447). The most common of these genome of which is known to be integrated in the host tumors are bronchiogenic carcinomas that secrete adreno- DNA) and leukemia in the adult AKR mouse (187). Both corticotrophic hormone. However, tumors of the thymus, increase concomitantly as a function of age (187), although kidney, and pancreas have also been shown to synthesize a it is not known whether the transformation to a leukemic variety of endocrine hormones. state allows the transcription of the viral genome or whether Other possible evidence of altered gene expression in the transcription of the viral sequences promotes the malig malignant states is the loss of expression of a2u-globulin [a nant condition. protein synthesized in the parenchymal cells of male rat The above discussions lead one to the conclusion that

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1978 American Association for Cancer Research. G. S. Stein et al. thÃ©normal cell contains the necessary information for the within the structural portion of a gene coding for a regula phenotypic expression of cancer. Even if one cannot fully tory molecule could result in the production of an incom accept that the manifestations of cancer are entirely the plete regulatory molecule. Also, the translocation and inter result of abnormalities in the control of genetic regulation, jection of genetic material between a gene-regulatory or one must certainly concede that malignant cells show the RNA polymerase attachment site and the structural portion derepression of fetal genes and express genes that are could adversely affect the regulation of the gene. normally expressed only in other cell types. These conclu Any abnormalities that result in a modified chromatin sions intimately relate the control of genetic expression structure also may be related to cancer. Recent work on with the disease state of cancer and strongly indicate that xeroderma pigmentosum patients, who have an extremely further study of regulatory mechanisms in eukaryotes will high incidence of skin cancer, has shown that an altered elucidate the mechanisms of neoplastic transformation. structure of chromatin is at least partially responsible for Mechanisms by Which Normal Control of Genetic the deficiencies in DNA repair in these patients (82, 85). Expression May Be Heritably Altered. The molecular Carcinogens are known to interact with DNA, protein, and mechanisms by which chemical carcinogens and radiation RNA (46, 176, 178, 197, 239, 280, 327, 354, 390, 448, 449, induce cancer are not well understood, but speculation and 464) and have been shown to modify chromatin structure correlative evidence (28, 463) have suggested modifications by inducing localized regions of DNA denaturation (127, leading to mutation of the cellular DMA. This modification 253, 464, 474). Duck reticulocyte DNA treated with N-2- could be either direct or indirect, such as alterations in acetylaminofluorene (a potent carcinogen) can be 60% molecules that decrease the fidelity of copying DMA or digested with the endonuclease S,, whereas control DNA interfere with DNA repair mechanisms. With the use of the can be digested only 5%. Although chromatin reconstituted final active metabolite (ultimate carcinogen) of many chem with A/-2-acetylaminofluorene-treated DNA and unmodified ical carcinogens, a good correlation has been established chromosomal proteins appears identical with control chro between mutagenesis and carcinogenesis (9, 10, 95, 177, matin with respect to susceptibility to digestion with 306). The following are means by which mutations could staphylococcal nuclease and nucleosome formation (474), result in heritable alterations in the control of genetic the modified reconstituted chromatin shows a 50% de expression. crease of in vitro RNA-synthesizing ability (474). The inhibi 1. Mutations of cellular DNA could conceivably occur in tion of RNA synthesis by aflatoxin B, (243) and by N- a portion of DNA coding for a regulatory protein. If the hydroxy-2-acetylaminofluorene (162, 211, 463, 485) has mutation occurred at the regulatory or RNA polymerase- been shown to be preferential for the 45S rRNA precursor. binding site, the gene could be rendered untranscribable. If Recent evidence on the study of benzo(a)pyrene-treated the mutation occurred in the structural portion, the result calf thymus DNA showed localized regions of denaturation, could be the production of a defective regulatory molecule. increased numbers of initiation sites for RNA polymerase, 2. If, as has been considered, regulatory molecules act and premature termination of transcription, presumably by direct binding to specific DNA sequences, thereby alter due to the interruption of the movement of the RNA polym ing the transcriptional ability of the sequence, mutation of erase along the template (329). Therefore it is evident that DNA could result in the inability of a regulatory molecule to carcinogens can modify chromatin structure and signifi recognize its binding site or alter the stability of such cantly affect the synthesis of cellular RNA. binding. Introduction of new genetic material into the cell, as in 3. Mutation of DNA could possibly result in the produc the case of viral episomes or integrated viral DNA, has been tion of defective molecules, such as histones or postsyn- extensively evaluated as a possible cause of cancer (29, thetic modifiers, that play an intermediary role in the rela 187, 188, 431, 432, 437). In human cancer, EBV and its relationship with Burkitt's lymphoma and nasopharyngeal tionship of a regulatory molecule and the genetic material. Chromosomal aberrations such as deletions, transloca carcinoma have received considerable attention. Support tions, or multiplicity of genetic material are caused by many ing a corelationship between EBV and cancer are the cancer-inducing agents such as radiation, certain muta- findings of Klein ef al. of EBV DNA and EBV-associated gens, and viruses (32, 112, 182, 291, 292, 370, 400, 452). nuclear antigens in human tumor cells (213, 223, 224, 257, Many persons with nonmalignant diseases associated with 295, 489). It is possible that included in the integrated viral gross chromosomal abnormalities (Fanconi's anemia, genome is the genetic information for a new regulatory Down's syndrome, Bloom's syndrome, ataxia-telangiecta- protein or an antagonist or modifier of host regulatory sia, XO, etc.) have been found to have a much higher molecules that results in altered genetic expression of the incidence of cancer, thereby suggesting a possible corre host DNA. Additionally, there exists the possibility that the lation between chromosomal abnormalities and malignant viral genome may be integrated in the host DNA in a manner diseases (13, 122, 134, 281, 282, 296, 484). A definite that sterically interferes with the regulatory and structural correlation has been established between the translocation segments of a gene. of a portion of Chromosome 22 to Chromosome 9 and As has been alluded to in the previous discussion, a chronic myelogenous leukemia (195, 297, 298, 335, 346). number of mechanisms that are consistent with current Chromosomal aberrations could result in alterations of the concepts for the etiology of cancer and that can heritably control of genetic regulation by the loss of genes coding alter normal control of genetic expression in eukaryotic for regulatory proteins or the translocation of structural cells are theoretically possible. Obviously, more informa genes, separating them from the DNA sequences at which tion as to the actual molecules involved in gene regulation regulation of the gene takes place. Translocation of DNA and their mode of action is required before more sophisti-

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1978 American Association for Cancer Research. Chromosomal Proteins and Gene Expression cated and accurate mechanisms for their alterations can be Some incidences of increased phosphorylation of H1 postulated. histone have been reported in rat hepatoma (19, 74); how Changes in Nuclear Proteins in Malignant Cells in Vivo. ever, Balhorn ef al. (17) observed that increased phospho If cancer is a biological situation in which normal control rylation of H1 histone in a variety of tumors could be directly mechanisms for genetic expression in eukaryotic cells are correlated with the degree of cell proliferation associated disastrously altered, then the logical question that comes with the tumor. Since H1 phosphorylation has been associ to mind is, "Can one observe any alterations accompanying ated with cellular replication (18, 20, 376), it is reasonable cancer in the molecules that are involved in the regulation to suspect that increased H1 phosphorylation in tumors is of genetic expression?" Having previously alluded to the related only to the increase in cell proliferation and is not roles that nuclear proteins play in this respect, let us unique in malignant diseases. examine the alterations in these proteins accompanying The relationship of alterations in histones to neoplastic malignant stages. However, caution should be exercised in transformation is not apparent at this time; however, it can interpreting results from such studies since neoplastic-as- be speculated that modifications of histones could result in sociated modifications in nuclear proteins may in part a destabilization of DNA:histone complexes or a favoring of reflect changes in the proliferative states of the cells in vivo histone:nonhistone protein complexes allowing for in as well as in vitro. creased availability of transcribable genes. Also carcino The histone fraction of the nuclear proteins, as has been gens have been shown by many investigators to bind discussed earlier, may be involved in nonspecific repres histones as well as NHCP and DNA (25, 104, 207, 288, 339, sion of genetic information and also in structural aspects of 369, 384, 461). In addition, it appears that carcinogens the genome. Differences in histones and in posttransla- preferentially bind to those chromatin subfractions that are tional modifications between neoplastic cells and their most active in transcriptional abilities (euchromatin) (288, normal counterparts have been found by Chromatographie 461). For example, a subfraction of mouse embryo cell and immunological techniques; however, in interpretation chromatin that represents 15% of the total chromatin and is of results concerning histones in normal and neoplastic twice as active in RNA synthesis as any other fraction binds cells, the possibility of NHCP contamination must be con 45% of the total 3-methylcholanthrene bound to chromatin sidered. It is known that some species of NHCP can be co- when 3-methylcholanthrene is present in the media of extracted with histones and coelectrophorese with the H1 growing cells (288, 461). The functional significance of fraction (49). Preparations of H1 histones contain a number carcinogen binding to nuclear components has, however, of closely related lysine-rich proteins and can be resolved not yet been resolved. by Chromatographie elution into several subfractions that Since components of the NHCP have been implicated as are characteristic of the source of H1 histones (58, 59, 90, the specific regulators of gene expression in mammalian 218, 314, 333, 386). Using these techniques, Kincade (219) cells, interest in the alterations of nuclear proteins in and Sluyser and Bustin (385) found that H1 histone subtrac malignant tissues has been directed towards the nonhis- tions from rat hepatoma have tumor-specific features in tone proteins. Parameters of these proteins that have been addition to features that are common to both normal and examined in neoplastic cells are NHCP:DNA ratios, fraction- malignant tissues. These investigators report that a sub- ation and comparison by 1- and 2-dimensional polyacryl- fraction of H1 found in normal rat liver either is absent or amide gel electrophoresis (53, 307, 480), immunological appears to be substantially diminished in hepatoma cells. competence of NHCP:DNA complexes (455), and posttran- These investigators also found, using microcomplement- scriptional modifications, such as phosphorylation. fixation techniques, that subfractions of H1 from rat hepa NHCP:DNA ratios have been shown to be significantly toma and rat liver are immunologically specific. It should be increased in hepatomas (11, 66, 72, 408) and human leuke kept in mind, however, that observed differences in sub- mia cells (91). When further examined by 1- or 2-dimen fractions of histones might reflect only differences in ex- sional polyacrylamide gel electrophoresis, the NHCP in tractability of the histone fractions rather than their ab experimental rat hepatomas (72, 307, 337, 408, 470, 481), sence. Indeed, differences in H1 histones do not seem to be mouse primitive teratocarcinoma cells (260), L-cells (260), characteristic of tumors, since Hohmann ef al. (181) could and neoplastic mammary cells of C3H mice and Fischer rats not detect any Chromatographie differences in H1 histone (208) showed qualitative and quantitative differences from fractions between mouse mammary tumors and normal their normal counterparts. The changes included proteins mammary gland tissue. Increases in certain histone methyl- found in normal liver and absent in hepatomas and proteins ation enzymes have been found in Novikoff hepatomas found in the hepatomas that could not be demonstrated in (313). However, certain carcinogens have been shown to normal liver (155, 156, 470). In other neoplastic systems inhibit histone methylation by methyltransferases (62). (480, 482), such as intestinal epithelial cells (39, 40) and Some investigators also suggest the possibility of increased human lymphocytes (479), differences in the NHCP of tumor histone-directed protease activity in cancer cells (111). and normal tissues have also been found. Studying changes DNA:histone ratios appear to be consistent with normal in NHCP of rat liver after exposure to diethylnitrosamine, controls in human leukemia (91) and experimental rat hepa Gronow and Thackrah (159) found a polypeptide with a tomas (408); however, differences in the relative quantities, molecular weight of 17,000 missing in the euchromatin of specific activities, and synthesis of histone fractions have treated cells. In contrast, no differences could be found in been reported in dimethylnitrosamine- and diethylnitros- the NHCP examined by polyacrylamide gel electrophoresis amine-induced rat hepatomas compared with normal rat between normal and azo dye-induced hepatomas (74). liver (154, 156, 408). Thiol components of nuclear proteins of rat liver after

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1978 American Association for Cancer Research. G. S. Stein et al. treatment with diethylnitrosamine and hepatoma 223 were electrophoresis. This result is in good agreement with the studied by Gronowef al. (153, 157, 160). The distribution of report of Ahmed (5) who noted a maximal incorporation of thiol components in the NHCP fraction of normal and 32Pinto NHCP 26 days after azo dye administration. Granner neoplastic tissues was similar but showed some quantita (149) also reported significant increases in protein kinase tive differences. Also the NHCP of hepatoma 223 were dif activity in HTC hepatoma cells. ferent from normal nonhistone proteins in their distribution In addition to reports of increased total nuclear protein when separated by the techniques of isoelectric focusing kinase activity in malignant cells (5, 74, 106, 149, 435), and sodium dodecyl sulfate gel electrophoresis (160). Ad some investigators have subdivided the nuclear protein ministration of 3-methylcholanthrene to rats has been re kinase activities by Chromatographie techniques and have ported to increase the incorporation of [14C]lysine into 2 M attempted further to characterize these enzymes in neoplas NaCI-extractable NHCP of the liver, and qualitative changes tic cells (106, 435). Farron-Furstenthal (106) reported the in the NHCP examined by disc electrophoresis or gel filtra separation by DEAE-cellulose of hepatoma nuclear protein tion were also noted between treated and nontreated livers kinase activity into 5 subfractions as compared with 3 (477). subfractions from normal liver nuclei. Thomson ef al. (435) That the NHCP fractions of certain tumor cells are not observed an additional protein kinase subfraction common identical with their normal counterparts is demonstrated by to Novikoff hepatoma, Ehrlich ascites, and Walker tumor immunological experiments (57, 478). Busch ef al. reported nuclei separated by phosphocellulose chromatography. finding a nuclear antigen in Novikoff hepatoma cells that This tumor-associated enzyme fraction had the unique formed precipitin bands in an immunoprecipitin assay with property of preferring Mn2+ to Mg2+ for optimal kinase chromatin proteins from Novikoff hepatoma, Walker 256 activity. This enzyme fraction was also responsible for the carcinosarcoma, and fetal rat liver. However, no precipitin phosphorylation of a single protein band (when phospho- bands were formed when chromatin proteins of normal or proteins were examined by polyacrylamide gel electropho regenerating rat liver were utilized (57, 478). Hnilica ef al. resis) that was found only in the neoplastic tissues, indicat (73, 455) reported that antiserum made from Novikoff hep ing that tumor cells possess not only a unique protein atoma DNA:NHCP complexes will only slightly react with kinase but also a unique phosphoprotein that is the sub DNA:NHCP complexes from rat liver and calf thymus. strate for the enzyme. DNA:NHCP complexes from other malignant tissues (AS- A few notes of caution should be introduced before one 30A hepatoma, Walker carcinosarcoma) cross-react with can seriously interpret the above-mentioned results con anti-hepatoma DNA:NHCP to a greater degree than normal cerning changes in nuclear proteins in normal and neoplas rat liver DNA:NHCP complexes. This suggests that more tic tissues. There exists a large difference in the proliferative similarity exists between NHCP of tumors of different origin state of normal and neoplastic tissues. In addition, during than between NHCP of malignant and normal counter malignant transformation, the original (or "normal") cell parts of the same cell type. Other investigators (64) have type is often replaced by a different, less differentiated cell immunologically examined tissues of tumor-bearing dogs type, and cases of chromosomal loss or addition are fre and have reported similar results. quent in neoplastic cell propagation. Also, the expression Also demonstrating differences in the NHCP components of benign endogenous viruses resulting in the production of normal and neoplastic tissues is the work of Kostraba of viral proteins is sometimes concomitant with malignant and Wang (233). These investigators demonstrated by RNA- transformation. Therefore the changes in nuclear protein DNA hybridization experiments the presence of RNA spe from normal to neoplastic tissues may simply be a reflection cies similar to those synthesized from Walker tumor when of the new proliferative state of the cell, the development of Walker tumor NHCP were used to activate (in vitro) tran a new cell type, differences in the chromosomal content, or scription of rat liver chromatin. Conversely, RNA species the expression of endogenous nontumorigenic virus and similar to those synthesized from rat liver chromatin were may not be directly related to the malignant transformation. demonstrated when NHCP from normal rat liver were used Obviously, it would be extremely informative if it were to activate in vitro transcription of Walker tumor chromatin. possible to identify or isolate cells that had been initiated in This work suggests that the NHCP are at least partially vivo by carcinogens but that were in a very early preneo- responsible for the transcriptional differences between rat plastic state. The cell type and chromosomal number of liver and Walker tumor. these early preneoplastic cells would be very close to their Phosphorylation of NHCP has been strongly implicated in normal state and variances of nuclear proteins would then the role of genetic regulation for reasons that have been be more likely to be related to the mechanisms involved in discussed previously. Hence alterations in phosphorylation the malignant transformation. The identification or isolation in cancer are especially interesting and have been studied of cells of this nature in vivo has up to now been restrictive; by many researchers (72, 74, 102). NHCP phosphorylation, however, recent reports offer the possibility of a new system specific activity with respect to 32P, and nuclear protein for the study of early preneoplastic and premalignant hep- kinase activity were found by ChiÃ¹ef al. (72, 74) to increase atocytes (392). This system is based on the principle that concomitantly with template activity in rat liver chromatin carcinogen-initiated hepatocytes have a relative resistance during /V,/V-dimethyl-p-(m-tolylazo)aniline administration. to the cytotoxic action of hepatocarcinogens and allows for Maximal protein kinase activity was observed 28 days after the proliferation of only these initiated cells. Perhaps with the continued introduction of the azo dye into the diet, and the development of systems such as this it will be possible at this time changes in the phosphorylation pattern of to study cancer-correlated changes in nuclear proteins with NHCP were evident when examined by polyacrylamide gel more validity.

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Changes in Nuclear Proteins in Transformed Cells. concluded that the differences in the 2 chromatins could be Since transformation of eukaryotic cells by viruses has attributed to the NHCP. Differences in chromatin from been related to the etiology of cancer (29, 187, 188, 431, normal and SV40-transformed fibroblasts have also been 432, 437), viral-transformed cells in culture offer a system detected by immunological techniques (487). Antisera made for studying biochemical and molecular alterations that from WI-38 fibroblast chromatin reacted only weakly with may elucidate the events occurring in vivo during malignant SV40-transformed WI-38 fibroblasts (2RA cell) chromatin. transformation. The study of cells in culture offers distinct An analogous result was found when 2RA chromatin anti- advantages. Unlike in vivo systems in which the tissues sera were reacted with WI-38 fibroblast chromatin. When studied may consist of several cell types, cell cultures allow the histone fractions of the chromatins were used as the for the study of homogeneous cell populations. Also, the antigens, the antisera reacted equally well with the histones environment of cells in culture can be completely con from WI-38 or 2RA cells. However, when NHCP fractions trolled, and the introduction of virus into the cell environ were used as the antigens, the heterologous NHCP fraction ment permits the study of the transformation process at all reacted with the antisera much less than did the original stages. Transformed cells have been extensively studied antigen. These differences could be explained either by and have been shown to exhibit the following properties: quantitative or qualitative differences in the NHCP fraction (a) a more rounded morphology (4); (to) an increased col of the 2 chromatins or by a different association or arrange ony-forming capability in agar (268); (c) a lower requirement ment of the NHCP in chromatin. for serum in the growth medium to obtain maximum cell More detailed studies of NHCP of normal and transformed growth (198); (d) increased lectin-initiated cell agglutination cells have provided further evidence for alterations in this (51); (e) a lower intracellular cyclic AMP concentration class of nuclear proteins accompanying viral transforma (309); (f) an increased plasminogen activator activity (308); tion. Variations in the rates of synthesis of NHCP in normal (g) an increased VmÂ¡,xfor2-deoxyglucose uptake (196); and compared with SV40-transformed fibroblasts have been (h) alterations of cell surface proteins (190). None of these observed (143, 192, 236, 248, 345), especially associated characteristics is absolute, and revertants exist that are with nonhistone proteins of high molecular weight transformed cells but that have lost 1 or more of the above (>100,000 daltons). This result is further supported by properties. Therefore, the most definitive criterion for clas reports of variations in NHCP synthesis after Rous sarcoma sifying a cell as transformed is based on its ability to form virus infection of chick embryo fibroblasts (412). Cholon tumors when injected into nude mice. and Studzinski (78) have suggested that the mechanisms Viral transformation of eukaryotic cells produces mor for NHCP synthesis in transformed cells may not be analo phological and biochemical changes that reflect alterations gous to those in normal WI-38 fibroblasts. These investiga in gene expression (33, 287, 430). In this regard, Fernandez- tors reported that the inhibition of low-molecular-weight Pol ef al. (109) have recently presented evidence that NHCP synthesis in WI-38 fibroblasts by aminonucleoside suggests that a-picolinic acid reversibly inhibits the growth cannot be demonstrated in SV40-transformed WI-38 fibro of cultured cells by blocking normal cells in the G, phase of blasts, suggesting that NHCP synthesis is in some way the cell cycle. However, transformed cells respond differ protected in the transformed cell. Differences in the com ently to a-picolinic acid, and the response is dependent on position of NHCP in normal and transformed cells as deter the transforming virus and independent of the cell line. For mined by examination of these proteins by the technique example, SV40-transformed cells are blocked in G-,, cells of polyacrylamide gel electrophoresis have been reported transformed by polyoma virus are blocked at both G, and by many investigators (235, 236). Also, evidence of in G.,, and cells transformed with Moloney virus are not creased turnover of these proteins has been established in blocked at any specific phase of the cell cycle after expo SV40-transformed cells (235). sure to a-picolinic acid. These results may indicate that a- Studies of posttranslational modification of NHCP in picolinic acid interferes with a specific growth control normal and transformed cells have indicated variations mechanism and that this control mechanism may be altered associated with transformation (235, 330). Increases in the in specific ways by different transforming viruses. Another phosphorylation of most molecular weight classes of NHCP striking example of abnormal gene expression is the acti in SV40-transformed WI-38 cells have been observed (330), vation of embryonic globin genes by Rous sarcoma virus in with a 10-fold increase in 32P incorporation into some chicken fibroblasts (161). Since nuclear proteins have been classes of nonhistone chromosomal phosphoproteins of implicated in structural and transcriptional properties of the SV40-transformed compared with normal WI-38 fibroblasts. genome, it is reasonable that changes in nuclear proteins This striking increase could not be explained by an in may accompany viral transformation and that such changes creased rate of synthesis of NHCP or an increased rate of may be related to transformation-associated modifications phosphate transport. Comparison of phosphorylation pat in gene expression. terns of NHCP by polyacrylamide gel electrophoresis has Evidence that substantial differences exist between chro- shown that there are specific effects on phosphorylation of matins from normal and transformed cells has been re individual protein species associated with viral transforma ported by Lin ef al. (256). These investigators detected tion (330). differences in the circular dichroism spectra of chromatins Alterations of the histones associated with viral transfor from normal and SV40-transformed WI-38 human fibro mation have not been reported to be as dramatic as those blasts and observed that these differences could be abol associated with the NHCP fraction. Studying normal and ished by prior washing of the chromatins with 0.25 M NaCI. SV40-transformed WI-38 fibroblasts, Krause and Stein (236) Since only NHCP are removed by 0.25 M NaCI, it was reported that, although all 5 major histone fractions were

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1978 American Association for Cancer Research. G. S. Stein et al. present in both cell populations, variations were found in System for the Detection and Classification of Mutagens and Carcino the relative amounts, rates of synthesis, and binding of gens. Proc. Nati. Acad. Sei. U. S., 70: 782-786, 1973. 11. Arnold, E. A., Buksas, M. M., and Young, K. E. A Comparative Study of histone fractions. This included an increase in the amount Some Properties of Chromatin from Two Minimal Deviation Hepato of histone H2A, an increase in the synthesis of histone H1, mas. Cancer Res., 33: 1169-1176, 1973. 12. Arosio, P., Adelman.T., Yokota, M.. and Drysdale, J. W. A Comparative and a decrease in the synthesis of histone H4 in transformed Study of Isoferritins in Normal and Malignant Tissues. In: W. H. cells. Nishimuraef al. (293) reported the presence of 2 new Fishman and S. Sell (eds.), Onco-Developmental Gene Expression, pp. 743-747. New York: Academic Press, Inc., 1976. histones on polyacrylamide gels after infection of BHK 13. Auerbach, A. D., and Wolman, S. R. Susceptibility of Fanconi's Anae cells with HSV-2, a virus associated with human cervical mia Fibroblasts to Chromosome Damage by Carcinogens. Nature, 267: carcinoma (336). Krause and Stein (237) have shown in 494-496, 1976. creased histone acetylation in SV40-transformed compared 14. Auerbach, R., Kubai, L., and Sidky, Y. Angiogenesis Induction by Tumors, Embryonic Tissues and Lymphocytes. Cancer Res., 36: 3435- with normal WI-38 cells. Similarly, Ledinko (247) has ob 3440, 1976. served increased histone acetylation in embryonic kidney 15. Axel, R., Cedar, H., and Felsenfeld, G. The Structure of the Globin Genes in Chromatin. Biochemistry, 14: 2489-2495, 1975. cells infected with adenovirus. Also histones are known to 16. Balhorn, R., Balhorn, M., and Chalkley, R. Lysine-Rich Histone Phos be contained within some DMA tumor viruses (123, 322), phorylation and Hyperplasia in the Developing Rat. Develop. Biol.. 29: and there is evidence that these viral-associated histones 199-203, 1972. 17. Balhorn, R., Balhorn, M., Morris, H. P., and Chalkley, R. Comparative are more highly acetylated than are host cell histones (362). High-Resolution Electrophoresis of Tumor Histones: Variation in Phos That acetylation of histones may be a significant factor in phorylation as a Function of Cell Replication Rate. Cancer Res., 32: viral transformation is proposed by the work of Schaffhau- 1775-1784, 1972. 18. Balhorn, R., Bordwell, J., Sellers, L., Granner, D. K., and Chalkley, R. sen and Benjamin (34, 362), who have correlated deficiency Histone Phosphorylation and DNA Synthesis Are Linked in Synchro of histone acetylation with loss of transforming ability of nous Cultures of HTC Cells. Biochem. Biophys. Res. Commun., 46. 1326-1333, 1972. polyoma virus. The significance of these observations is not 19. Balhorn, R., Chalkley, R., and Granner, D. Lysine-Rich Histone Phos known at this time. phorylation: A Positive Correlation with Cell Replication. Biochemistry, 11: 1094-1098, 1972. Prospects 20. Balhorn, R., Reike, W. O., and Chalkley, R. Rapid Electrophoretic Analysis for Histone Phosphorylation. A Reinvestigation of Phosphoryl While an important role for chromosomal proteins in ation of Lysine-Rich Histone during Rat Liver Regeneration. Biochem dictating structural and functional properties of the eukar- istry, 10: 3952-3958, 1971. 21. Baliga, B. S., Borek, E., Weinstein, l. B., and Srinivasan, P. R. yotic genome is apparent, much remains to be resolved Differences in the Transfer RNAs of Normal Liver and Novikoff Hepa- before gene regulation in eukaryotic cells can be under toma. Proc. Nati. Acad. Sei. U. S., 62: 899-905, 1969. 22. Balinsky, D., Cayanis, E., and Bersohn, E. Comparative Kinetic Study stood. Predicated on recent developments in the area of of Human Pyruvate Kinases Isolated from Adult and Fetal Livers and chromosomal protein research, conceptual as well as tech from Hepatoma. Biochemistry, 12: 863-870, 1973. nological, it is reasonable to anticipate that significant 23. Balinsky, D., Cayanis, E., Geddes, E. W., and Bersohn, I. Activities and Isoenzyme Patterns of Some Enzymes of Glucose Metabolism in Hu progress can be expected towards comprehending the man Primary Malignant Hepatoma. Cancer Res., 33: 249-255, 1973. manner in which expression of specific genes is governed. 24. Barrett, T., Maryanka, D., Hamlyn, P., and Gould, H. Nonhistone Proteins Control Gene Expression in Reconstituted Chromatin. Proc. Resolution of this problem will undoubtedly provide insight Nati. Acad. Sei. U. S., 71: 5057-5061, 1974. into the cellular and molecular basis of a broad spectrum of 25. Barry, E. J., Ovechka, C. A., and Gutmann, H. R. Interaction of Aromatic Amines with Rat Liver Proteins in Vivo. II. Binding of W-2- disease processes, particularly neoplasia, in which aberra Fluorenylacetamide-9-'4C to Nuclear Proteins. J. Biol. Chem., 243. 51- tions in gene expression appear to be a primary component 60, 1968. of the etiology. 26. Bartley, J., and Chalkley, R. Further Studies of a Thymus Nucleohis- tone-Associated Protease. J. Biol. Chem., 245. 4286-4292, 1970. 27. Baserga, R. Nonhistone Chromosomal Proteins in Normal and Abnor References mal Growth. Life Sci., 75: 1057-1071, 1974. 1. Abelev, G. I. Antigenic Structure of Chemically-Induced Hepatomas. 28. Becker, F. F. Cancer: A Comprehensive Treatise, Vol. 1. Etiology: Progr. Exptl. Tumor Res., 7: 104-157, 1965. Chemical and Physical Carcinogenesis. New York: Plenum Press, 1975. 2. Abelev, G.I. Alpha-Fetoprotein in Ontogenesis and Its Association with 29. Becker, F. F. Cancer: A Comprehensive Treatise, Vol. 2. Etiology: Viral Malignant Tumors. Advan. 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Gary S. Stein, Janet L. Stein and Judith A. Thomson

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