Chromosomal translocations with specific breakpoints in asbestos-induced rat mesotheliomas.

Cytogenetic analysis was conducted on cells of 15 rat mesotheliomas induced in rats by the i.p. inoculation of crocidolite or chrysotile asbestos. These tumor cells were diploid, triploid, or tetraploid. All tumor lines exhibited aneuploidy and marker chromosomes. Loss of at least one copy of the X chromosome was observed in each of the tumors analyzed, and loss of copies of chromosomes 8, 16, 20, or 18 characterized at least six of the tumors. Translocations were observed in 12 tumors, with six chromosome rearrangements present in at least two different tumors. However, the breakpoints were not always identical. On the other hand, translocations involving chromosomes 5, 10, and 13 exhibited repeated breakage at the same loci. Such specific and repetitive translocations may be involved in the process of asbestos-induced tumor development.

Induction of DNA strand breaks in cultured rat embryo cells by crocidolite asbestos as assessed by nick translation.

Asbestos, a proven carcinogen, is reported to have no genotoxic effects. We hypothesized, however, in light of its clastogenic effects that one mechanism by which asbestos induces cell transformation and tumorigenesis involves the induction of DNA strand scission. Cultured rat embryo cells were exposed to low concentrations of International Union Against Cancer crocidolite and examined at intervals ranging from 2 to 48 h. The induction of DNA strand breaks was examined using the technique of nick translation followed by autoradiography or scintillation counting. Our results indicate that cells exposed to crocidolite have a higher incidence of DNA breaks and that this effect becomes apparent within 2-6 hours of exposure. Ball-milled crocidolite as well as riebeckite have a significantly lower effect while glass fibers induce a more pronounced DNA strand damage. These observations support the role fiber length plays in carcinogenesis and suggest that the classification of asbestos as a nongenotoxic carcinogen be reconsidered.

Nutritional folate-deficiency in Chinese hamster ovary cells. Chromosomal abnormalities associated with perturbations in nucleic acid precursors.

Folate deficiency is known to induce chromosomal abnormalities. We used a nutritionally folate-deficient Chinese hamster ovary (CHO) cell culture system to examine modulation of chromosome damage by purine or pyrimidine supplementation. The cells were cultured in folate-deficient (Fol-) medium or Fol- medium supplemented with thymidine (dT) or hypoxanthine (Hx) until population growth arrest. The cultures were then switched to complete medium, permitting the cells to begin cell division. Cell-cycle progression was followed by flow cytometry to identify the first mitosis, when samples for analysis were collected. The mitotic index, frequency of chromosomal aberrations in mitotic cells, and relative distribution of different types of aberrations were determined. Cells grown in Fol- medium supplemented with Hx entered the G2/M phase of the cell cycle at 14 hours after media change as compared with 16 hours for Fol- cultures or 24 hours for Fol- cultures supplemented with dT. Cells cultured in Fol- medium alone or supplemented with dT showed similar frequencies of damage, averaging 20-22%, as compared with 2% for control cultures. In contrast, cells grown in Hx-supplemented medium exhibited a lower frequency of damaged mitoses (15%), as well as a reduction in certain types of abnormalities. This latter finding is surprising in light of our previous work showing that the presence of Hx during folate deficiency produced a more severe perturbation of phenotype (cellular enlargement) and growth control (S-phase delay and cell killing) than did dT supplementation or the absence of both nucleotide precursors.

Molecular analyses of in vivo hypoxanthine-guanine phosphoribosyltransferase mutations in human T-lymphocytes: II. Demonstration of a clonal amplification of hprt mutant T-lymphocytes in vivo.

Recent molecular analysis of in vivo-derived hprt mutant T-lymphocytes cloned from human blood show that mutants occurring at the normal frequency (approximately 5 X 10(-6) in healthy young individuals generally represent independent hprt mutations. Here we report that in an individual with a high mutant frequency (86-620 X 10(-6],92% (61/66) of the mutant clones are descendents of an original mature T-cell precursor that has undergone in vivo clonal expansion. Therefore, these mutants could represent as few as one original hprt mutation. If so, correcting for the clonal expansion yields a revised calculated mutant frequency (Mf) value for this individual that is near the normal range. These hprt mutant clones all showed identically rearranged T-cell receptor (TCR) beta and gamma gene patterns by Southern blot analysis. All the clones were surface marker CD4+, showed no obvious chromosomal aberration, and had no detectable hprt gene structural alteration. This TCR-defined T-cell clone appears to have expanded in the blood of the individual over a 6-month period and persists at high levels after nearly 4 years. This finding illustrates the need to analyze mutants from individuals with high mutant frequencies at the molecular level in order to estimate hprt mutation frequency from the calculated hprt mutant frequency. The possibility that spontaneous hprt mutants might arise in vivo preferentially in dividing cells, and implications of this, are discussed.

Changes in S-phase associated with differentiation of mouse embryos in culture from blastocyst to early somite stage.

Mouse embryos, cultured for 8 days from the blastocyst (stage 5 of Theiler, 1972) to the 10--12 somite stage (stage 13) were labeled at daily intervals with 5 muCi/ml 3H-thymidine for 2 h, sectioned, and processed for autoradiography. The frequency of labeled cells, as well as the intensity of labeling, exhibited a progressive decline during the period of growth of embryos in culture. Endoderm and ectoderm cells were characterized by significant differences in the labelling index and the rate of DNA syntheses. These changes indicate that, during mammalian embryogenesis, the relative duration of the S-phase declines and that the absolute duration of the S-period expands.

Acute exposure of female hamsters to carbendazim (MBC) during meiosis results in aneuploid oocytes with subsequent arrest of embryonic cleavage and implantation.

A single oral dose of the fungicide and microtubule poison, MBC, administered to female hamsters at proestrus, results in infertility and early pregnancy loss (1). To characterize the site and mode of action of this effect, direct assessments of oocyte chromosomes, fertilization, and preimplantation embryo development were made. Female hamsters were given a single dose of MBC (1,000 mg/kg) on the afternoon of proestrus (to coincide with meiotic maturation of the oocytes) and either killed shortly after ovulation (day 1) to recover oocytes, or bred and killed on gestation day (gd) 1 to 5 of pregnancy to assess fertilization and preimplantation embryo development and enumerate early implantation sites. Chromosome analysis in unfertilized oocytes revealed an MBC-induced increase in aneuploidy (37 vs. 14% in controls). When animals were bred after dosing, MBC had no effect on the number of oocytes recovered or fertilized. However, significant increases were found in the proportion of embryos that failed to reach the expected stage of development, namely, the eight-cell stage on the afternoon of gd 3, the morula stage by the morning of gd 4, and the blastocyst stage by the afternoon of gd 4 (a time when some embryos have implanted). The mean number of implantation sites, revealed by Evans Blue staining, was also significantly lower in treated females on the afternoon of gd 4 and the morning of gd 5. These simple direct assessments elucidated a mechanism of MBC-induced early pregnancy loss, induction of aneuploidy in oocytes. They also ruled out an effect on fertilization, but demonstrated a subsequent arrest of preimplantation embryonic development accompained by a decrease in the likelihood of implantation.

Incidence of chromosome aberrations in mammalian sperm stained with Hoechst 33342 and UV-laser irradiated during flow sorting.

The separation of two sperm populations is possible using the technique of flow sorting, provided that a significant difference exists in the DNA content of X- and Y-bearing sperm. In order to ascertain whether or not chromosome damage was induced in sorted sperm, chromosome preparations were made from isolated sperm that had been microinjected into hamster eggs. While egg chromosomes exhibited a low frequency of chromosome aberrations, ranging from 4 to 7%, a large proportion of sperm cells exhibited chromosome damage. Between 29% of unstained and unsorted sperm and 38% of stained and unsorted sperm exhibited some type of chromosomal abnormality and this proportion increased to 50% in sorted sperm. If only damaged sperm nuclei are considered, the two unsorted sperm groups had a mean of 0.6 breaks, 0.8 triradial exchanges, and 0.2 quadriradial exchanges per nucleus. However, sorted sperm, which were stained with a fluorochrome and exposed to UV-laser irradiation, exhibited a mean of 2.9 breaks, 2.6 triradial, and 1.9 quadriradial exchanges per nucleus in which damage occurred. These observations indicate that the treatments and manipulations to which sperm nuclei are subjected during flow sorting cause chromosomal aberrations, and that exposure of the cells to UV-laser irradiation contributes substantially to the chromosome damage observed.

The ordered arrangement of chromosomes in the Chinese hamster spermatocyte nucleus.

The question of chromosome distribution in the mammalian nucleus is addressed, and data are provided in support of the ordered arrangement of chromosomes in the Chinese hamster spermatocyte. Testicular cells were dispersed and air-dried without prior fixation, then stained and karyotyped. The position of chromosome telomeres in 217 pachytene spermatocytes was determined in relation to four concentric rings which equally divided the nuclear area. The distribution of telomeres showed a progressive decline from the central to the peripheral rings. This was particularly pronounced for chromosomes 1-7, but was reversed for the XY chromosomes. The distribution of the total as well as of the individual chromosomes was significantly different from that expected on the basis of random distribution. The only exceptions to this were chromosomes 8-10, which exhibited random distribution. Thus, while chromosomes 1-7 had a central position, the XY pair had a peripheral localization. The mean ring position appeared to be related to chromosome length, except for the XY chromosomes, suggesting that chromosome length may determine chromosome position.

The creeping vole, Microtus oregoni: karyotype and sex-chromosome differences between two geographical populations.

The G-banded karyotype of the creeping vole, Microtus oregoni, prepared from animals trapped in Oregon and Washington, is presented. The two populations had similar autosomal banding patterns but exhibited striking differences in their sex chromosomes. The X chromosome of voles captured in Oregon was 39% longer than that of voles trapped in Washington. The length difference was primarily due to an increase in size of light G-bands, which, in both populations, comprised large segments of the X chromosome. On C-banding, the X chromosome exhibited major blocks of constitutive heterochromatin corresponding to the light G-bands. In contrast, the Y chromosome of the Oregon voles was 24% shorter than that of the Washington voles and lacked the short arm and some terminal bands present in the Washington voles.

Gonadotrophin-induced reinitiation of meiosis in testes of oestradiol-treated prepubertal rats.

Male rats injected with 50 micrograms oestradiol dipropionate every 5 days from birth were given daily injections of 20 i.u. PMSG from 20 to 29 days of age. Testicular weight and cell number remained relatively constant in rats treated only with oestradiol and were significantly below normal levels, but significantly increased in PMSG-treated animals. Dissociated cells from PMSG-treated rats incorported 560% as much [3H]thymidine as did cells from oestradiol-treated animals at 21 days, and 240% at 29 days. The increase was due to new cells entering the S-phase, as indicated by an increase in the labelling index. Autoradiographic examination of testicular tissue indicated that PMSG stimulated DNA synthesis in meiotic and somatic cells. The number of labelled germinal cells increased and numerous pachytene spermatocytes were observed after 6--9 days of treatment. Myoid and interstitial cells were markedly stimulated by PMSG. The presence of spermatids in 26- and 29-day-old PMSG-treated animals indicated recue of pachytene spermatocytes destined to degenerate after oestradiol administration. It is concluded that the hormonal control of the meiotic process is similar in prepubertal and adult rats.

Analysis of the progression of meiosis in dispersed rat testicular cells by flow cytofluorometry.

Initiation and progression of meiosis was followed in dispersed rat testicular cells by flow cytofluorometry and cytology. The DNA content of dissociated testicular cells of rats 6--30 days old, killed at daily intervals, was analysed by flow cytofluorometry using propidium iodide as a DNA-specific and quantitative fluorochrome. Testicular cells of a 6-day-old rat showed one peak of fluorescence. A second peak, at twice the modal channel number, appeared in testicular cells of 9-day-old animals. The number of cells under this peak increased progressively with age. A third peak, at half the channel number of the original one, appeared at 20 days and accounted for an increasing proportion of cells in testes taken from older rats. Cytological examination of the testicular tissue used for flow cytofluorometric analysis showed that preleptotene spermatocytes first appeared at 8 days after birth. Spermatids were first observed cytologically at 20 days after birth. The close temporal appearance of the fluorescence peaks with that of spermatocytes and spermatids, and the close association of the frequency of diploid and tetraploid cells as derived by flow cytofluorometry and cytology, indicated that the fluorescence peaks correspond--in order of increasing fluorescence--to spermatids, spermatogonia and somatic cells, and to spermatocytes. This conclusion was re-examined by analysing the ploidy levels of testicular cells of hypophysectomized or estradiol-treated by flow cytofluorodmetry. There was a loss of the haploid and tetraploid peaks subsequent to hypophysectomy. Estradiol dipropionate-treated rats, given weekly injections starting at 7 days of age, showed no appearance of the haploid peak and the regression of the tetraploid peak after an initial and transitory appearance. These results indicate that changes in ploidy levels that accompany the progression of meiosis in the testis were reflected in the sequential appearance of three fluorescence peaks as detected by flow cytofluorometry. The close correlation between the frequency of cell types as obtained by cytology and flow cytofluorometry indicates that the latter is a sensitive method for studying selected aspects of spermatogenesis in dissociated testicular cells.

Sequential development and tissue organization in whole mouse embryos cultured from blastocyst to early somite stage.

The development of mouse embryos in culture from the implantation to the head-fold stage was sequentially examined. Our goal was to compare the morphology of embryos grown in vitro to those developed in vivo, published in standard texts, and to delineate the stages involved in the process of tissue differentiation and organization. Mouse blastocysts (stage 6) were collected at 3.5 days p.c. and cultured. Attachment of the blastocysts occurred on the second day of culture (stage 8). Following the collapse of the blastocyst endoderm cells began to migrate and to encircle the inner cell mass. At 2 days in culture the embryonic and extra-embryonic ectoderm became distinguishable and the proamniotic cavity appeared (stage 9). Egg cylinders began to project above the substrate at 2.5 days in culture (stage 10) and to progress through the stages observed in vivo. At 4 days a posterior amniotic fold began to form (stage 11) and was followed at 5 days by the formation of the chorion, the appearance of mesoderm, exocoelom, and head fold (stage 12). At 6 days in culture the embryo had differentiated longitudinally and developed an allantois, blood islands, Reichert's membrane, head process, and primitive streak. At 7 days somites as well as the neural fold and heart were observed (stage 14) and were followed by further differentiation at 8 days (stage 15). These observations indicate that apparently normal embryo development can be maintained in vitro through the early stages of organogenesis, thus providing a unique opportunity for investigating the regulation of early mammalian development.

Ultrastructural changes induced by steroids and gonadotropins in cultured rat Sertoli cells.

Testicular cells readily grew in cultures of seminiferous tubule fragments derived from adult rat testes. They formed confluent monolayers and continued to grow through four passages. The majority of cells had ultrastructural features characteristic of Sertoli cells. They exhibited large nuclei lacking heterochromatin, having nuclear membrane clefts, prominent nucleoli, significant amounts of agranular endoplasmic reticulum, large and numerous mitochondria, and lipid droplets. Cultured Sertoli cells responded to follicle-stimulating hormone (FSH), androgens, and dbcAMP by undergoing distinct morphological and ultrastructural changes. FSH induced parallel arrays of granular endoplasmic reticulum. Testosterone and dbcAMP resulted in the swelling of mitochondria, the formation of parallel cristae that stretched across the lumen, and the appearance of lipid droplets. Sertoli cells, cultured in a medium containing testosterone and FSH since their initiation, developed extensive agranular endoplasmic reticulum, strikingly large lipid droplets, and mitochondria that were characterized by either a dark matrix and vesicular cristae, or a light matrix and parallel cristae. These results indicate that Sertoli cells respond in vitro to hormones that are active in vivo, and support the suggestion that the Sertoli cell is a site of steroid synthesis in the mammalian testis.

Characteristics of tumors and tumor cells cultured from experimental asbestos-induced mesotheliomas in rats.

Mesotheliomas developed in rats in the abdominal cavity 6-23 months after peritoneal introduction of chrysotile and crocidolite asbestos. The tumors were strikingly similar to those occurring in man both with regard to histologic features and growth patterns. The authors have cultured cells from these tumors and established epithelial lines with a variety of karyologic features and doubling times shorter than those of normal mesothelial cells. Lines of diploid or near-diploid tumor cells required serum in the medium to replicate, whereas most aneuploid cell lines were maintained in a serum-free medium. In serum-free medium, the aneuploid line monolayers produced anchorage-independent excrescent masses of cells which "bud" and float free. These spheroids, which strikingly resemble the papillary structures in human mesotheliomas, are composed of mesothelial-like cells that produce hyaluronic acid and have a rich complement of intermediate filaments, predominantly cytokeratins. Aneuploid cells also replicated in soft agar with high efficiency, whereas the diploid and near-diploid cells did not. All but one cultured cell line, regardless of karyotype, produced tumors after subcutaneous or intraperitoneal inoculation (or both). Aneuploid cells caused lung tumors sporadically when introduced intravenously. Comparative analysis of the nuclear DNA of primary tumors and cultured cells demonstrated a high degree of chromosomal instability and selection among cells during early passage in vitro.

Cytogenetic analysis of malignant skin tumors induced in chemically treated TG-AC transgenic mice.

TG.AC mice (which carry a v-Ha-ras transgene) rapidly develop papillomas in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). Approximately 30% of the papillomas are associated with subsequent development of malignancies. Early-passage spindle-shaped tumor cells arising from explant cultures of TPA-induced tumors in TG.AC mice were tumorigenic when transplanted to syngeneic recipients. The v-Ha-ras transgene in the transplanted tumors was expressed at a high level. To identify possible genetic changes associated with the development of malignant tumors, explanted cells were cultured in vitro and assessed for karyotypic changes between the second and third passages by analyzing G-banded metaphase chromosomes. For comparison, skin malignancies were induced in nontransgenic FVB/N mice (parent strain) by 7,12-dimethylbenz[a]anthracene (DMBA) initiation and TPA promotion, and their G-banded metaphase chromosomes were analyzed. Trisomy (in at least 50% of about 30 metaphases) of chromosome 15 (in five of 15 tumors) and chromosome 6 (four of 15 tumors) was observed in TG.AC mice, independent of chemical treatment or tumor type. Of six tumors from DMBA/TPA-treated FVB/N mice, three had trisomy 10 or 15 (or both), and two appeared normal. The absence of trisomy 7 is notable because c-Ha-ras maps to that chromosome. The absence of trisomy 7 in the six FVB/N DMBA/TPA-induced skin malignancies contrasts with DMBA/TPA-induced karyotypic effects in SENCAR mice. Expression of the v-Ha-ras transgene may have precluded the requirement for endogenous mutant ras and allelic imbalance involving chromosome 7 in TG.AC mice, but it could not have in FVB/N mice. These results suggest the possibility that the observed trisomies are consequential, rather than causal, events in the development of TG.AC or FVB/N skin malignancies. Molecular genetic analysis will be required to understand the changes associated with tumorigenesis in this transgenic line as well as in the parent mouse line.