Spontaneous and induced aneuploidy,considerations which may influence chromosome malsegregation

Aneuploidy plays a major role in the production of human birth defects and is becoming increasingly recognised as a critical event in the etiology of a wide range of human cancers. Thus, the detection of aneuploidy and the characterisation of the mechanisms which lead to chromosome malsegregation is an important area of genotoxicological research. As an aid to aneuploidy research, methods have been developed to analyse the mechanisms of chromosome malsegregation and to investigate the role of aneuploidy in tumour progression. The presence of aneuploid cells is a common characteristic of many of tumour cell types as illustrated by the wide range of chromosome number changes detected in post-menopausal breast tumours. To investigate the time of occurrence of aneuploidy during tumour progression, we have studied the chromosome number status of Syrian hamster dermal (SHD) cells cultures progressing to morphological transformation. The production of both polyploid and aneuploid cells is a common feature of progressing cells in this model. The elevation of both progression to morphological transformation and aneuploid frequencies can be produced by exposure to a diverse range of carcinogens and tumour promoters. Analysis of the genotoxic activity of the hormone 17-beta oestradiol demonstrated its ability to induce both chromosome loss and non-disjunction in human lymphoblastoid cells implicating aneugenic activity in hormone related cancers. Mutations in the p53 tumour suppressor gene introduced into human fibroblasts produced modifications in chromosome separation at mitosis which may lead to the production of both aneuploidy and polyploid cells. Our studies indicate that the production of aneuploid cells can be influenced by both endogenous and exogenous factors and occur throughout the progression of normal cells to a malignant phenotype.     

Characterization of an in vitro micronucleus assay with Syrian hamster embryo fibroblasts

The use of Syrian hamster embryo cells for assessing genotoxicity provides the unique opportunity to determine 5 different end-points (gene mutations, DNA-strand breaks, aneuploidy, DNA repair (unscheduled DNA synthesis, UDS) and neoplastic transformation) in the one cell system. This approach allows direct comparisons of results produced under identical conditions of dose at target, metabolism and bioavailability. We report here on the characterization of an additional end-point in the same cell system: the formation of micronuclei indicating chromosomal changes induced by chemicals. For a preliminary validation of this new test system we have investigated 14 carcinogens and 3 non-carcinogenic structural analogues in order to evaluate the significance of micronucleus induction for carcinogenic properties. All tested carcinogens induced micronuclei in a dose-dependent manner; all non-carcinogens yielded negative results. Correlations between the formation of micronuclei and the Ames test, induction of UDS, cell transformation and the in vivo bone marrow micronucleus test are demonstrated.     

Mitotic inhibition and aneuploidy induction by naturally occurring and synthetic estrogens in Chinese hamster cells in vitro

We used a predominantly diploid Chinese hamster cell line to test a number of naturally occurring and synthetic estrogens for their ability to arrest cells at metaphase, their potential for allowing anaphase recovery, and their capability of inducing aneuploid progeny. The chemicals employed included diethylstilbestrol, dienestrol, hexestrol, beta-estradiol, ethynylestradiol and estriol. We also tested progesterone, estrone and testosterone in this regard. Only estrogens and their synthetic analogs caused mitotic arrest and aneuploidy, while progesterone, estrone and testosterone did not cause mitotic disturbances. Among the estrogens, DES was the most effective arrestant on a comparative molar basis, whereas dienestrol was most potent over a wide range of concentrations. Estriol was the least potent as an arrestant but was an effective inducer of aneuploidy. The addition of a metabolic activator (S9) did not alter the ability of DES to arrest mitosis. Following the removal of the drugs, cells were able to quickly reorganize a spindle apparatus and enter anaphase. Diethylstilbestrol, dienestrol, hexestrol, beta-estradiol, ethynylestradiol and estriol caused significant increase in aneuploidy within a narrow range of high concentrations in recovering cell populations. Aneuploidy was induced in a non-random manner. Immunofluorescence studies with anti-tubulin antibody indicate that estrogens may have a mechanism of mitotic arrest similar to that of colchicine and colcemid, viz inhibiting the polymerization of tubulin to form microtubules. These data suggest that the interaction between estrogens and microtubules may mediate the induction of aneuploidy in somatic cells. Aneuploidy induction by DES and similar compounds may be related to their carcinogenic potential.     

Colchicine-like effect of diethylstilbestrol (DES) on mammalian cells in vitro

Diethylstilbestrol (DES), a synthetic estrogen, showed colchicine-like effects in vitro on cells of the cell lines such as Chinese hamster fibroblast of thymus origin (CHT), rat liver (DL), rat erythroblastic leukemia (EDEN-1/TC) and HeLa-S3. Metaphase arrest was induced 3 h after treatment with 15 microgram/ml of DES and polyploid or polynucleated cells were prominently observed more than 24 h after treatment. The arrest, however, was reversible when the agent was removed from the medium. Tetraploid karotypes induced by DES in CHT cells consisted of all double sets of diploid chromosome constitution except one chromosome marker. By clonal selection, several hypotetraploid sublines were successfully isolated from a CHT cell population after the treatment with 15 microgram/ml of DES for 48 h. Some comparative studies of cytological effects of DES with those induced by colcemid indicated that the DES effect was also a mitotic inhibition similar to colchicine.     

The induction of aneuploidy by nitrogen mustard in a human lymphoblastoid cell line

We report that the presence of an extra Y chromosome can be used as a marker for the induction of aneuploidy (mitotic non-disjunction) in a human lymphoblastoid cell line. This endpoint is easily visualized in metaphase chromosome preparations after staining with quinacrine mustard. The induction of cells with two Y chromosomes by nitrogen mustard (NM) was examined. Exposure to 150 ng/ml nitrogen mustard induced a 6-fold increase in aneuploid frequency relative to untreated control levels; maximal induction of aneuploidy was observed 2 days after treatment. Lower concentrations of nitrogen mustard (36 and 75 ng/ml) induced smaller increases in aneuploid frequency, with maximal induction observed 1 day after treatment. This system has the potential to be used as an assay for the induction of aneuploidy in cultured human cells.     

Examining the link between chromosomal instability and aneuploidy in human cells

Solid tumors can be highly aneuploid and many display high rates of chromosome missegregation in a phenomenon called chromosomal instability (CIN). In principle, aneuploidy is the consequence of CIN, but the relationship between CIN and aneuploidy has not been clearly defined. In this study, we use live cell imaging and clonal cell analyses to evaluate the fidelity of chromosome segregation in chromosomally stable and unstable human cells. We show that improper microtubule-chromosome attachment (merotely) is a cause of chromosome missegregation in unstable cells and that increasing chromosome missegregation rates by elevating merotely during consecutive mitoses generates CIN in otherwise stable, near-diploid cells. However, chromosome missegregation compromises the proliferation of diploid cells, indicating that phenotypic changes that permit the propagation of nondiploid cells must combine with elevated chromosome missegregation rates to generate aneuploid cells with CIN.     

Aneuploidy, the primary cause of the multilateral genomic instability of neoplastic and preneoplastic cells

Cancers have a clonal origin, yet their chromosomes and genes are non-clonal or heterogeneous due to an inherent genomic instability. However, the cause of this genomic instability is still debated. One theory postulates that mutations in genes that are involved in DNA repair and in chromosome segregation are the primary causes of this instability. But there are neither consistent correlations nor is there functional proof for the mutation theory. Here we propose aneuploidy, an abnormal number of chromosomes, as the primary cause of the genomic instability of neoplastic and preneoplastic cells. Aneuploidy destabilizes the karyotype and thus the species, independent of mutation, because it corrupts highly conserved teams of proteins that segregate, synthesize and repair chromosomes. Likewise it destabilizes genes. The theory explains 12 of 12 specific features of genomic instability: (1) Mutagenic and non-mutagenic carcinogens induce genomic instability via aneuploidy. (2) Aneuploidy coincides and segregates with preneoplastic and neoplastic genomic instability. (3) Phenotypes of genomically unstable cells change and even revert at high rates, compared to those of diploid cells, via aneuploidy-catalyzed chromosome rearrangements. (4) Idiosyncratic features of cancers, like immortality and drug-resistance, derive from subspecies within the 'polyphyletic' diversity of individual cancers. (5) Instability is proportional to the degree of aneuploidy. (6) Multilateral chromosomal and genetic instabilities typically coincide, because aneuploidy corrupts multiple targets simultaneously. (7) Gene mutation is common, but neither consistent nor clonal in cancer cells as predicted by the aneuploidy theory. (8) Cancers fall into a near-diploid (2 N) class of low instability, a near 1.5 N class of high instability, or a near 3 N class of very high instability, because aneuploid fitness is maximized either by minimally unstable karyotypes or by maximally unstable, but adaptable karyotypes. (9) Dominant phenotypes, because of aneuploid genotypes. (10) Uncertain developmental phenotypes of Down and other aneuploidy syndromes, because supply-sensitive, diploid programs are destabilized by products from aneuploid genes supplied at abnormal concentrations; the maternal age-bias for Down's would reflect age-dependent defects of the spindle apparatus of oocytes. (11) Non-selective phenotypes, e.g., metastasis, because of linkage with selective phenotypes on the same chromosomes. (12) The target, induction of genomic instability, is several 1000-fold bigger than gene mutation, because it is entire chromosomes. The mutation theory explains only a few of these features. We conclude that the transition of stable diploid to unstable aneuploid cell species is the primary cause of preneoplastic and neoplastic genomic instability and of cancer, and that mutations are secondary.     

Neoplastic conversion of preneoplastic Syrian hamster cells: rate estimation by fluctuation analysis.

Analysis of the role of gene mutations in the multistep process of neoplastic transformation requires that the discrete steps in carcinogenesis first be dissected. Toward this end, we have isolated and characterized preneoplastic Syrian hamster cells which exhibit in vitro a trait highly correlated with neoplastic conversion in vivo. Previous findings (J. C. Barrett, Cancer Res. 40:91-94, 1980) indicate that spontaneous neoplastic transformation of Syrian hamster cells occurs in at least two steps. An intermediate stage, characterized by an aneuploid established cell line which has a propensity to become neoplastic spontaneously upon further growth in vitro, has been described. These preneoplastic cells differ from diploid early-passage Syrian hamster cells in becoming capable of anchorage-independent growth in semisolid agar, as well as becoming neoplastic in vivo when attached to a solid substrate. Evidence presented here demonstrates that anchorage-independent conversion in vitro is a reliable marker for neoplastic conversion in this cell system. Fluctuation analyses, patterned after those described by Luria and Delbruck for microbial genetics, demonstrate that anchorage-independent variants are generated randomly from clonally derived preneoplastic cells at the rate of 10(-8) to 10(-7) variants per cell per generation. These results establish a multistep stochastic process for transformation in vitro and indicate that conversion to anchorage independence may be necessary for Syrian hamster cells to become tumorigenic. The possible role of gene mutation in this step during neoplastic progression is discussed.     

Induction of aneuploidy by mitotic arrestants in mouse bone marrow

Most human and animal carcinogens induce gene mutation, chromosome breakage or other types of DNA lesions. However, recent studies indicate that some carcinogens do not directly damage DNA, but may cause missegregation of chromosomes resulting in aneuploidy production. Aneuploidy-producing agents pose serious genetic hazards to the human population. Such agents may cause genomic imbalance not only in somatic cells which may result in cancer development, but also in germinal cells which may result in the production of abnormal offspring (e.g. Down's syndrome). To limit human exposure to potential aneuploidy-producing agents, such agents must first be identified in experimental animals. The present study demonstrates that vinblastine and colcemid are capable of inducing aneuploidy in bone marrow cells of treated mice. Both of these compounds are chemotherapeutic agents that arrest mitosis by interfering with the formation of spindle microtubules. Single intraperitoneal injections of vinblastine, at a dose of 9 mg/kg, were found to produce 1.5-5.2% of hyperdiploidy in all of the 10 treated mice sampled at 17-96 h after injection. Only the frequency of hyperdiploidy was determined because hypodiploid cells could result from artifactual chromosome loss during slide preparation. At 0.9 mg/kg, vinblastine was found to produce 0.5-3.5% of hyperdiploidy in 8 of the 10 treated animals. The frequency of hyperdiploid cells in animals treated with colcemid was low. A dose as high as 37 mg/kg was found to produce only 0.5-1% of hyperdiploidy in 3 of the 10 treated animals, and hyperdiploidy was observed only in animals sampled at 17-24 h. In 10 mice treated with saline alone, no hyperdiploid cells were observed. Unlike cell cultures where vinblastine and colcemid had been shown to be equally effective in producing aneuploidy, vinblastine was found in this study to be a much more potent aneuploidy inducer than colcemid in mice.     

Cytogenetic analysis of Q-banded pronuclear chromosomes in fertilized Syrian hamster eggs

The frequency and type of chromosome abnormalities were analyzed in 917 female pronuclei in Syrian hamster eggs fertilized by human sperm. Analysis at this stage allows detection of errors which have occurred during meiosis I and II. The chromosomes were Q-banded to identify individual chromosomes and detect subtle alterations. Thirty-three (3.6%) of the hamster egg complements were abnormal: 19 (2.1%) were hypohaploid, seven (0.76%) were hyperhaploid, two (0.2%) had double aneuploidy, and five (0.5%) had a structural chromosome abnormality. Since there were significantly more hypohaploid than hyperhaploid complements, a conservative estimate of aneuploidy can be derived by doubling the frequency of hyperhaploid complements. Thus a minimal estimate of aneuploidy (single, 1.5%, and double, 0.2%) is 1.7% and a minimal estimate of the total frequency of abnormalities is 2.2%. All chromosome groups were represented among the aneuploid complements suggesting that all chromosomes are susceptible to non-disjunction.     

Neoplastic transformation of cultured Syrian hamster embryo cells by DNA of herpes simplex virus type 2.

Syrian hamster embryo cells were transformed to a neoplastic phenotype after exposure to herpes simplex virus type 2 (S-1) DNA at concentrations (less than or equal to 0.01 microgram per 60-mm dish) at which infectivity was no longer demonstrable. Transformed cells manifested in vitro phenotypic properties characteristic of the neoplastic state, expressed herpes simplex virus-specific antigens, and induced invasive tumors in vivo. Transfection and transformation of Syrian hamster embryo cells with herpes simplex virus type 2 DNA or its fragments is a suitable system for investigating the structure and function of herpes simplex virus-transforming gene(s).     

Induction of unscheduled DNA synthesis and micronucleus formation in Syrian hamster embryo fibroblasts treated with cysteine S-conjugates of chlorinated hydrocarbons

S-(chloroethyl)-cysteine (CEC) and S-(1,2-dichlorovinyl)cysteine (DCVO) have been proposed as intermediates in the metabolic transformation of the carcinogens 1,2-dichloroethane and 1,1,2-trichloroethylene. We have tested the ability of CEC and DCVC to induce DNA repair and genotoxic effects at the chromosomal level by comparative assessment of unscheduled DNA synthesis induction and micronucleus formation in Syrian hamster embryo fibroblasts. CEC induced a potent and dose-dependent response in both assays, whereas DCVC treatment resulted in a comparatively weak induction of DNA repair and failed to raise micronucleus formation above control rates. Inhibition of cysteine conjugate \gB-lyase diminished the effect of DCVC, but had no influence on the genotoxicity of CEC either in the unscheduled DNA synthesis or micronucleus assay.Abbreviations AOAA aminooxyacetic acid - CEC S-(chloroethyl)-cysteine; \gB-lyase, cysteine conjugate -lyase - DCE 1,2-dichloroethane - DCVC S(1,2-dichlorovinyl)-cysteine - GSH glutathione - HU hydroxyurea - IBR IBR-modified Dulbecco's Eagle's reinforced medium - MN2 micronuclei/2,000 cells - 4-NQO 4-nitroquinoline-1-oxide - SHE Syrian hamster embryo fibroblasts; ³H-Thd, ³H-thymidine - TCE 1,1,2-trichloroethylene - UDS unscheduled DNA synthesis     

Induction by modified purines (2-aminopurine and 6-N-hydroxylaminopurine) of chromosome aberrations and aneuploidy in Syrian hamster embryo cells

The modified purines, 2-aminopurine and 6-N-hydroxylaminopurine, are known point mutagens in prokaryotic organisms. 2-Aminopurine is much less potent than 6-N-hydroxylaminopurine in inducing gene mutation in mammalian cells in culture and this corresponds to the relative activity of these two compounds in inducing tumors in rats and neoplastic transformation of Syrian hamster embryo cells in culture. We report here that these modified purines can induce chromosome aberrations, including chromatid gaps, breaks, and exchanges, as well as numerical chromosome changes in Syrian hamster embryo cells. These chromosome mutations occur over the concentration range of chemical needed to induced morphological transformation of the same cells. It is not known how nucleic base analogs induce chromosome mutations; however, this activity must be considered in attempting to understand the mechanism by which these agents induce neoplastic transformation of cells.     

A genetic assay for aneuploidy: quantitation of chromosome loss using a mouse/human monochromosomal hybrid cell line

A genetic assay is described in which a mouse/human hybrid cell line R3-5 containing a single human chromosome (a monochromosomal hybrid) is used to detect chemically induced aneuploidy. In this assay the frequency of chromosome loss determined by the cloning efficiency of the cells in a selection medium is used as an index for the potential of a chemical to induce aneuploidy. The hybrid cells are deficient in hypoxanthine guanine phosphoribosyltransferase (HGPRT) and contain human chromosome 2, marked with Ecogpt, an E. coli gene for xanthine guanine phosphoribosyltransferase. These cells with a genotype of hgprt-/Ecogpt+ can grow in medium containing mycophenolic acid and xanthine (MX medium) but not in medium containing 6-thioguanine (6-TG). The loss of the human chromosome from R3-5 cells as a result of chemical treatment produces cells with a genotype of hgprt-/Ecogpt- which are capable of growth in the medium containing 6-TG. Thus, the cloning efficiency of cells treated with a test chemical in 6-TG provides a method to determine the frequency of cells that have lost the human chromosome. Two chemicals, colcemid and nocodazole, previously known to induce aneuploidy in mammalian cells were used for a preliminary evaluation of this test system. Both of these compounds at concentrations ranging from 0.002 to 0.032 micrograms/ml showed a concentration-related positive response in this assay.     

Inability of diethylstilbestrol to induce 6-thioguanine-resistant mutants and to inhibit metabolic cooperation of V79 Chinese hamster cells

The mutagenicity of diethylstilbestrol (DES) in V79 Chinese hamster cells was examined under a variety of conditions. DES over a concentration range 0.01–10 μg/ml failed to induce any increase above the spontaneous frequency of 6-thioguanine-resistant V79 cells. The effect of varying the expression time after treatment in the mutation assay from 3 to 9 days was studied and DES was nonmutagenic at all time points, while N-methyl-N′-nitro-N-nitrosoguanidine was highly mutagenic with a peak response after a 5–7 day expression time. The mutagenicity of benzo[a]pyrene and DES, both of which induce morphological and neoplastic transformation of Syrian hamster embryo (SHE) cells, was tested by cocultivating V79 cells with SHE cells for possible metabolic activation of the chemicals. Neither compound was mutagenic to V79 cells in the absence of SHE cells. Benzo[a]pyrene, but not DES, was mutagenic to V79 cells cocultivated with SHE cells. These results support the observation that DES can induce cell transformation under conditions that do not result in any measurable gene mutations. Moreover, the ability of DES to enhance the recovery of 6-thioguanine-resistant mutations was studied by determining the ability of DES to inhibit metabolic cooperation of V79 cells. Unlike the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate, DES was a weak or inactive inhibitor of metabolic cooperation.     

In vitro induction of numerical changes in the karyotype of normal and transformed Djungarian and Chinese hamster cells]

The comparative study of the frequency of colcemid-induced aneuploidy and polyploidy in cultured normal and transformed cells of Djungarian hamster is described. The occurrence of variants with changed chromosome number is much higher in populations of SV40-transformed cell line (4/21) than in normal embryonic cultures. In transformed lines of Djungarian and Chinese hamsters (4/21 and V-79) the frequency of cells with changed chromosome number was found to be dependent on the culture density: the percentage of polyploids was 4-5-fold higher when the number of seeded cells was 2-fold lower. The highest number (18-29%) of hypermodal cells was produced at drug concentrations of 0.02-0.025 mkg/ml. The percengate of polyploids under these conditions reached 10-20. At further increase of colcemid concentrations the proportion of polyploid cells increased. In Djungarian hamster embryonic cell cultures there were single cells with changed chromosome numbers at a concentration of the drug of 0.015-0.1 mkg/ml.     

Occurrence of sister-chromatid exchange and chromosomal aberration during vitamin A-induced cell differentiation in vitro

Prompted by the recent growth in interest in the mechanisms of vitamin A (VA) action, we studied the effects of VA on the frequency of sister-chromatid exchange (SCE) and chromosome aberration (CA) in a culture system using a fetal Syrian hamster (female) pulmonary epithelial cell line (M3E3/C3). When manipulated by specific culture conditions, the cells in this system could be rendered competent for activation of polycyclic aromatic hydrocarbons. Cells induced to such a state were exposed to 0, 2, 8 and 24 micrograms/ml of VA for 4 days. The average frequency of SCE per metaphase increased from 1.64 at 0 micrograms/ml to 3.44 at 24 micrograms/ml with a moderate degree of dose dependence. In addition, the q-terminal area of X-chromosomes appears to be one of the most specifically vulnerable sites for SCE due to VA. The frequency of CA encompassing triradial, quadriradial, quinqueradial, ring and dicentric chromosomes also increased in a rather sigmoid fashion from 3.6% at 0 micrograms/ml to 14.8% at 24 micrograms/ml. Apart from the frequently demonstrated protective roles or otherwise less often encountered promotional effects of VA in the development of squamous metaplasia, neoplasia, neoplastic transformation or mutation, an alternative interpretation for the current results implies a possible relationship between SCE and CA caused by VA and cell differentiation and/or drug resistance mechanisms.     

Dislocation of chromatin elements in prophase induced by diethylstilbestrol: a novel mechanism by which micronuclei can arise

The in vitro micronucleus test with Syrian hamster embryo (SHE) cells assays the induction of micronuclei by chemical agents. Both chromosome fragments and lagging chromosomes can give rise to micronuclei. Nevertheless, only limited information is available on the ultrastructure of micronuclei and the mechanisms of their formation. Diethylstilbestrol (DES), a non-mutagenic carcinogen, as well as its analogue 3.3'-DES induce micronuclei in SHE cells. A comparison of the dose response of DES-induced micronucleus formation with the previously published ones for aneuploidy and transformation shows that all 3 run in parallel. Thus, a functional relationship between these endpoints, in the SHE system, may be implied. The present study is designed to address the formation of micronuclei using supravital UV microscopy, to test for the presence of defined chromosome domains within micronuclei using immunocytochemistry, and to define aspects of their ultrastructure by electron microscopy. Supravital UV microscopy showed that 3.3'-DES induces displacement of chromosomes/chromatids during prophase/anaphase and formation of micronuclei during cytokinesis. Immunocytochemistry revealed that micronuclei contain, at high frequencies, CREST antibody-reactive kinetochores, indicating the presence of whole chromosomes or centric fragments in these structures. Moreover, transmission electron microscopy showed that micronuclei exhibit ultrastructural details typical of interphase nuclei. Specifically, micronuclei exhibited morphological evidence of a nuclear lamina and segregation of karyoplasm into euchromatic and heterochromatic regions. All micronuclei examined were enclosed by a nuclear envelope of normal morphology and showed nuclear pore complexes. Together the findings provide evidence that DES interferes with the mitotic apparatus as early as prophase, resulting in the formation of micronuclei and, as a consequence, in the loss of chromatids or chromosomes.     

Mutagenic effect of BUdR in diploid human fibroblasts

It has only recently been possible to demonstrate the expected mutagenic effect of 5-bromodeoxyuridine (BUdR) in heteroploid hamster cells in culture. We have now extended this observation to diploid human fibroblasts utilizing techniques adapted from the work of Albertini and DeMars on X-ray mutagenesis at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus in these cells. In four separate experiments, fibroblasts from a female donor were exposed to 500 micrograms/ml ethylmethane sulfonate (EMS) or 3 micrograms/ml BUdR yielding survivals of 9% and 5%, respectively. After a 6-day expression period, survivors were plated in selection medium containing 0.3 micrograms/ml 8-azaguanine (8-AG). After 3-5 weeks, azaguanine-resistant colonies were isolated for characterization or stained for counting. The average spontaneous mutation rate/cell/generation was 0.6.10(-6). The average induced mutation rates for EMS and BUdR were 7.8.10(-6) and 6.3.10(-6)/cell/generation, respectively. Similar results were obtained in two experiments with an additional fibroblast line. Mutant colonies isolated following BUdR treatment demonstrated from 1.4 to 61.5% of the HGPRT activity of the parental line and showed at least 8% Barr bodies, excluding the possibility of contamination by Lesch-Nyhan cells. This demonstration of a BUdR effect comparable to that of an alkylating agent or X-irradiation opens the study of mutation due to base-analog substitution in diploid human cells.     

Rodent cell transformation assays-a brief historical perspective

In vitro cell transformation is a process characterized by a series of progressive distinctive events that often emulate manifestations occurring in vivo and which are associated with neoplasia. Attendant cellular and sub-cellular alterations include, among others: cellular immortality, phenotypic changes, aneuploidy, genetic variability, cellular disarray, anchorage-independent growth, and tumorigenicity in vivo. Early chemically induced neoplastic transformation studies involved the use of normal diploid (Syrian) hamster embryo (SHE) cells and monitored the formation of morphologically altered colonies. Later investigations employed primarily two established mouse cell lines, i.e. the BALB/c 3T3 A31 cell line and the C3H 10T 1/2 cell line, and monitored the induction of morphologically aberrant foci. In either case, such transformed cellular clusters (colonies and foci) could induce tumors upon inoculation in vivo. Some subsequent noteworthy advancements using these systems included pH adjustments, metabolic supplementation, amplification of expression of formerly latent transformed foci, concurrent detection of mutagenesis and transformation, and use of a Bhas 42 cell line (v-Ha-ras transfected BALB/c 3T3 cells) to detect both tumor initiators and promoters. Over time, such transformation assay systems have been found useful in academic, industry and regulatory laboratories, generally for research purposes, but also occasionally as screening tools for potential chemical carcinogens. Nevertheless, to date, use of these assays for decision-making purposes in the regulatory arena remains elusive and will require comprehensive validation to gain universal acceptance.