The effect of partially purified thrombopoietin on guinea pig megakaryocyte ploidy in vitro.

Enriched populations of guinea pig bone marrow megakaryocytes were prepared by density gradient and velocity centrifugation and maintained in liquid cultures for 24 or 48 h. The resulting megakaryocyte preparations were of 86.1% +/- 5.5% purity. After 24 or 48 h in liquid culture, recovery of viable cells was 77% +/- 11% or 83% +/- 13%, respectively. Megakaryocyte cultures were supplemented with 100-200 micrograms/ml of either a lectin-fractionated preparation of thrombopoietin (TPO) from the plasma of thrombocytopenic rabbits or an identically prepared protein fraction from non-thrombocytopenic animals. Addition of TPO resulted in a significant increase (p less than 0.05) in both the proportion and total numbers of 32N megakaryocytes and a significant decrease (p less than 0.05) in the relative frequency of 8N megakaryocytes. In most experiments, a decrease in the total number of 8N megakaryocytes also was noted. These results indicate that partially purified TPO is able to increase the ploidy (DNA levels) of megakaryocytes in vitro.

The in vitro growth response of primary human colorectal and gastric cancer cells to gastrin.

A series of 31 colorectal and 13 gastric primary human tumours were screened for their growth response to human gastrin-17 in vitro, as assessed by 75Se-seleno-methionine incorporation. Fifty-five percent of colorectal and 69% of gastric tumours showed a significant trophic response to the hormone. The responses were achieved at physiological gastrin concentrations (post-prandial circulating gastrin levels) in 35% of colorectal and 55% of gastric tumours. Lymphocytes from tumour-associated lymph nodes showed no response to the hormone and "normal" mucosal cells (obtained from the resection margin of the surgical specimen) showed lower mean levels of 75Se-seleno-methionine uptake (colorectal: 110%; gastric: 119%, expressed as a percentage of the control) when compared to tumours (colorectal: 151%; gastric: 147%). The small number of well differentiated and/or Dukes' stage A colorectal tumours examined were gastrin-responsive, but all the responsive gastric tumours were poorly differentiated. With respect to ploidy, 89% of diploid and 67% of aneuploid colorectal tumours responded trophically to gastrin. Patients with colorectal or gastric tumours may benefit from treatment with gastrin antagonists.

[Evaluation of testicular damage by flow cytometry: testicular atrophy caused by ethylene oxide].

The new method using flow cytometry was applied to analyse the testicular toxicity of ethylene oxide, and the usefulness of this method is discussed. When Wistar male rats were exposed to ethylene oxide for six hours a day, three times a week for six weeks, the testicular weights of the exposed group significantly decreased. When the cells of these testes were stained by propidium iodide and analysed by flow cytometry, four peaks which corresponded to maturation phase spermatids (less than C), the other haploid cells (C), diploid cells (2C) and tetraploid cells (4C) were obtained. Calculating the ratio of the percentage of less than C, C and 4C to that of 2C, the ratio of less than C of the exposed group decreased by 72.9%, 2C by 53.5% and 4C by 5.1% when compared with the control group. As these changes were almost consistent with that of histopathological examinations, we are able to conclude that more mature germ cells were affected by ethylene oxide. This method by flow cytometry is thought to be objective, quantitative and convenient to evaluate testicular damage by chemicals.

Sequential cytopunctures during preoperative chemotherapy for primary breast carcinoma. Cytomorphologic changes, initial tumor ploidy, and tumor regression.

Thirty-five patients with large but operable breast carcinoma (T3, N0-N1, M0) received before surgery three cycles of preoperative Adriamycin (doxorubicin), vincristine, cyclophosphamide, methotrexate, 5-fluorouracil (AVCMF) chemotherapy (CT). All patients had sequential cytopunctures for both cytologic examination and flow cytometric DNA analysis (FCM): one before treatment and the three others after each cycle of CT. At cytologic examination, 20 carcinomas showed CT-induced cytomorphologic changes. These changes in malignant cells were also evaluated on histologic sections after surgery. A significant relationship was found between cytomorphologic changes in cytopunctures and in the corresponding tumor tissue. The lobular carcinomas did not reveal changes either on cytologic or on histologic study. At FCM analysis before treatment, ten carcinomas were diploid and 25 were nondiploid. When initial tumor ploidy was compared to cytomorphologic changes, none of the diploid carcinomas showed changes. An objective tumor regression was observed in 12 of 20 tumors with cytomorphologic changes and in four of 15 tumors without changes on cytologic examination. But, a significant relationship appeared only when cellular changes were evaluated on histologic analysis. When tumor regression was compared to ploidy before treatment, the rate of objective regression was significantly higher in nondiploid tumors (15/25) than in diploid tumors (one of ten). From these findings, initial tumor diploidy could be a predictor of tumor chemoresistance, whereas cytomorphologic changes during chemotherapy could be an indicator of tumor cell chemosensitivity.

The influence of perimenopausal age on estrogen receptor status and estradiol and DNA content in human breast cancer.

In 25 cases of breast cancer occurring in perimenopausal age, estrogen receptor (ER) content was determined by the dextran-coated charcoal (DCC) assay and both endogenous-bound estradiol and nuclear DNA concentrations were measured by computerized quantitative analysis on formalin-fixed paraffin-embedded tissue samples. No statistically significant relationship (p greater than 0.05) was found within these parameters. The high incidence (64%) of ER-negative cases in this menopausal age was mainly due (62.5%) to interference of high levels of endogenous estradiol occupying the receptor sites in-vivo. The prevalence of hormone-insensitive and aneuploid cell clones accounted for the remaining true ER-negative tumours (37.5%).

Chromosomal and c-K-ras oncogene alterations induced by a chemical carcinogen and phorbol ester in skin fibroblasts of individuals with familial polyposis coli.

Skin fibroblasts derived from three patients with familial polyposis coli (FPC) were treated in vitro with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) alone or in combination with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). None of the cultures treated four times with MNNG alone (1 microgram/ml) or in combination with TPA (eight applications, 0.1 microgram/ml each) showed either morphological transformation or anchorage-independent growth for 18 months after treatments. FPC cells treated with MNNG alone showed cell growth inhibition, breakage and loss of chromosomes, as well as the deletion of 5.7 kilobase (kb) EcoRI fragment in the c-K-ras locus as detected by Southern blot analysis with v-K-ras specific probe (clone KBE-2). The control cells contained two EcoRI fragments of 5.7 and 4.2 kb. On the other hand, cells treated with MNNG first and then followed by treatment with TPA not only showed an increase in the rate of cell growth but also exhibited two novel EcoRI fragments of 9.4 and 12 kb. Treatment with TPA alone appeared to stimulate cell division long after application and to induce chromosomal aberrations but had no effect on c-K-ras sequences. The most likely explanation for the appearance of chromosome pulverization and hyperploidy in FPC cells treated with TPA is the induction of premature chromosome condensation of the S phase cells due to fusion with the mitotic chromosomes.

The influence of phenobarbital and butylated hydroxytoluene on the ploidy rate in rat hepatocarcinogenesis.

The effect of the 'promoters' phenobarbital (PB) and butylated hydroxytoluene (BHT) on the ploidy changes during hepatocarcinogenesis in rats was compared in a densitometric analysis of Feulgen-stained nuclei on paraffin-embedded tissue slices. The triphasic Gerlans protocol for liver-cancer induction was applied. Initiation with a single dose of diethylnitrosamine (DEN), and selection with 2-acetylamino-fluorene (2-AAF) combined with a proliferative stimulus (CCl4 administration), was followed by a treatment with PB or BHT for periods up to 22 weeks. Control animals received no treatment after the initiation and selection procedure. Despite intra- and inter-individual variations, an increase in the amount of 2N nuclei is found in the putative preneoplastic lesions of animals that received initiation and selection (I-S) and 3 weeks basal diet (BD). When the diet is supplemented with PB (after I-S), the increase of diploid nuclei starts earlier. At the time carcinoma arise (22 weeks PB treatment) a decrease in the frequency of 2N nuclei is found. BHT-treated animals which develop no carcinoma within the considered timespan, show a clear increased amount of 2N nuclei in the precancerous lesions only after 14 weeks treatment. It seems that there is a positive correlation between the outgrowth of putative preneoplastic foci and nodules in rat liver and an increase of diploid nuclei in these lesions. PB, as promoter used after initiation and selection, speeds up the development of carcinoma in rat liver, and therefore also the shift to diploidization in these rats starts earlier in comparison with I-S-treated rats. Although BHT does not promote liver carcinogenesis, an increase of diploid nuclei is also observed here during lesion formation. It may, therefore, be concluded that the phenomenon of diploidization is closely linked to and probably necessary for preneoplastic development, but that it is not an absolute indicator for neoplastic transformation.

[Effect of a food ration with a low-protein content on the processes of liver parenchyma recovery in rats after tetrachloromethane damage]. / Vliianie pishchevogo ratsiona s nizkim soderzhaniem belka na protsessy vosstanovleniia parenkhimy pecheni krys posle povrezhdeniia tetrachlormetanom.

Male Wistar rats weighing 190-200 g were fed a low protein diet. Atrophy of cytoplasm was observed after a 3-week malnutrition. In early periods of liver regeneration (6 hr after CCl4 poisoning), an increase in DNA synthesis was accompanied by an increase in summary concentration of organelle membranes. Though their ploidy was higher than in the control, volume of hepatocytes and summary area of organelles surface membrane was smaller than in standard diet. These data show a reduced capacity for realization of genetic program during the regeneration period in protein-deficient rats.

Hypoploidy and hyperplasia in the developing brain exposed to alcohol in utero.

Prenatal effects of acute maternal alcohol ingestion on chromosome segregation and mitotic frequency in the brain cells of the fetus were evaluated in mice by direct chromosome and mitotic counts and by flow cytometry. Fetuses were exposed to acute transplacental doses of alcohol for 4 days and killed on the fifth day. Those litters in which the fetuses were developed to the equivalent of normal 16th-17th-day gestation age were analyzed. A marked increase in the number of hypoploid metaphases was observed in direct proportion to the dose ingested by the mother. An over 30% increase in hypoploidy over controls was measured in the fetuses exposed to the highest dose. Counts of mitotic cells showed an over tenfold increase in the mitotic index of the fetal brain exposed to alcohol. Flow cytometric measurements of DNA content in isolated fetal brain cell nuclei showed a shift from a single G0/G1 peak in controls to a bimodal G0/G1-G2 + M distribution in alcohol-exposed fetuses of the same developmental age.

Metaphase and anaphase analysis of V79 cells exposed to erionite, UICC chrysotile and UICC crocidolite.

The cytogenetic effects of erionite treatment of V79 cells were compared with those of UICC crocidolite and UICC chrysotile treatment. A significant reduction in diploid cells with an accompanying increase in aneuploid and polyploid cells was observed with all three treatments. In the erionite-treated cultures, an increase in aneuploidy was observed at all dose levels ranging from 10 to 100 micrograms/ml, whereas in the crocidolite- and chrysotile-treated cultures, significant increases in aneuploidy were observed at all dose levels except the low dose, 10 micrograms/ml. Chromatid aberrations were observed in cultures treated with crocidolite and chrysotile and were especially pronounced at dose 100 micrograms/ml of chrysotile. The clastogenic effect of erionite was weaker but statistically significant at dose 100 micrograms/ml. An extrapolation of these cytogenetic changes over dose in number of fibers suggests that erionite was more reactive than the other two minerals in producing aneuploidy. The number of fibers required to produce a similar degree of cytogenetic effects was several orders of magnitude higher for chrysotile and crocidolite than erionite. These results correlate with the higher tumorigenic potency of erionite. In general, fewer cells treated with erionite entered anaphase than those treated with the other two minerals. As a result, abnormal anaphases representing chromosomal mis-segregation were observed only in the chrysotile- and crocidolite-treated cultures. To our knowledge, this is the first report on cytogenetic effects of erionite.

High yield of micronuclei and micronuclei premature chromosome condensation in a mouse tumor cell line cultured in vivo with prearrested mitotic metaphases.

It is generally argued that micronuclei and micronuclei premature chromosome condensation (MN PCC) in stable cell lines occur only after treatment with potent clastogens, mutagens or carcinogens. In the present paper we report on the occurrence of micronuclei and MN PCC (predominantly of S type) at a high frequency in a mixed in vivo culture of prearrested mitotic metaphases with an asynchronous population of mouse ascites tumor cells (S180) without the aid of any clastogen, mutagen or carcinogen.

Chromosome aberrations induced by 9-aminoacridine derivatives.

The induction of chromosomal aberrations by 5 derivatives of nitro-9-aminoacridine in V79 Chinese hamster cells was observed. The clastogenic activity of the compounds tested depended on the position of the NO2 group in the acridine ring. The strongest clastogens were derivatives with NO2 in position 1. The remaining derivatives placed in decreasing order of clastogenic activity were: 3-nitro, 4-nitro and 2-nitro. In addition, 2-nitro and 3-nitro derivatives produced hyperdiploid/polyploid metaphases.

Megakaryocyte DNA content and platelet formation in rats after a sublethal dose of thio-TEPA.

The megakaryocytopoiesis in rats was studied following a single dose of thio-TEPA to determine the mechanisms for the thrombocytopenia and subsequent recovery. The blood platelet number, platelet production (measured by 35S incorporation into platelets), mean platelet volume, and the number and DNA content of bone marrow megakaryocytes (MK) were observed. The blood platelet counts, platelet production, and total MK number decreased to low values following the administration of thio-TEPA, and stayed low until regeneration started around day 10. The mean platelet volume increased from the normal 6.6 fl to 7.8 fl during the early regeneration days 8-14. The MK were divided into four ploidy classes: 2N-4N, 8N, 16N, and 32N-64N. The number of MK within all ploidy classes decreased to nearly zero within four days after the injection of thio-TEPA. The regeneration started around day 10 with increasing numbers of 2N-4N and 8N MK, while the number of 16N and 32N-64N MK increased more than four days later. It is concluded that decreased or blocked influx of progenitor cells into the MK compartment is the main reason for thrombocytopenia after exposure to thio-TEPA.

In vitro binding of mirex by mouse hepatocytes.

Mirex (dodecachlorooctahydro-1,3,4-metheno-2H-cyclobuta[cd]+ ++pentalene) is a hepatic tumorigen that is shown to cause marked disturbances in hepatic cell ploidy in rodents. Kinetic measurements of [14C]mirex binding were performed in freshly prepared diploid (DP) and polyploid (PP) hepatocytes, as well as in erythrocytes, under controlled conditions. The binding of mirex to hepatocytes, irrespective of their ploidy, was partially Na+-dependent and totally Ca2+-independent. Variations in temperature and pH appeared to significantly inhibit mirex binding; the optimum binding was seen at 37 degrees C under physiological pH. The saturation kinetic data revealed that PP cells were saturated at a very low concentration of mirex (two- to threefold) compared to DP, exhibiting a high-affinity binding of mirex to PP with a low Km (347 nM) and Vmax (102 nmol/mg X min). The Km (550 nM) and Vmax (340 nmol/mg X min) values determined for DP cells were of higher magnitude, like those of erythrocytes (Km, 819 nM; Vmax, 330 nmol/mg X min), indicating that distinct differences exist in the binding affinities of three cell types. However, erythrocytes and DP cells showed close similarity in their Vmax values. Interestingly, mirex levels in the lipid compartments of DP and PP cells revealed no apparent differences. The results are discussed in terms of the possible susceptibility of PP cells and their role in the initiation of toxic response leading to hepatotumorigenesis in rodents.

Irreversible depression in the ratio of tetraploid:diploid liver nuclei in rats treated with 3'-methyl-4-dimethylaminoazobenzene (3'M).

A reduction in the ratio of tetraploid to diploid liver nuclei has been investigated as an early indicator of hepatocarcinogenesis in the rat using the liver carcinogen 3'-methyl-4-dimethylaminoazobenzene (3'M). In a dose ranging study 3'M was administered by gavage to rats at 5, 12.5 and 25 mg/kg for up to 10 weeks and the following parameters studied: bodyweight gain, dye binding to hepatic protein, nuclear ploidy in liver and histopathology. Significant reduction in bodyweight occurred only with 25 mg/kg; dye binding to protein occurred in a dose-related manner; depression of the percentage of tetraploid nuclei compared with diploids was dose-related and effects were detected even at the lowest dose. These observations were consistent with those from previous studies by other investigators. In a separate experiment 3'M was administered at the maximum tolerated dose (MTD) of 25 mg/kg for 3 weeks, during which time there was a significant reduction in bodyweight gain and a reduction in the ratio of tetraploid:diploid liver nuclei. After cessation of dosing the rate of bodyweight gain returned to normal but there was no corresponding recovery of the ratio of tetraploid:diploid nuclei in the liver. A long-term continuous gavage study at 2.5 mg/kg revealed a time dependent reduction in the ratio of tetraploid:diploid liver hepatocyte nuclei and histopathological changes that included hepatocarcinoma were also observed. There was no correlation between the severity of pathological changes and the change in nuclear ploidy ratio in this experiment and it is concluded that the changes in ploidy ratio are related to the carcinogenic effect of 3'M and are independent of its gross toxicity.

Nuclear ploidy of neonatal rat livers: effects of two hepatic carcinogens (mirex and dimethylnitrosamine).

The effect of two hepatic carcinogens, dimethylnitrosamine (DMN) (genotoxic) and mirex (epigenetic), on polyploidization in 12-d-old neonatal rats was investigated by Coulter counteranalysis and [3H] thymodine uptake in isolated hepatic nuclear classes. DMN disturbed the normal ploidy development in the neonatal liver and the proportion of nuclei in the ploidy classes by inducing the premature formation of a significant population of tetraploids with a concomitant reduction in diploids. A great proportion of the replicative activity was present in tetraploid nuclei as measured by the incorporation of [3H] thymidine. The labeling index and number of mitoses were also increased. In contrast to DMN, mirex had no influence on polyploidization. The neonatal rats used in these studies thus offer an opportunity to investigate in vivo the mode of action of genotoxic versus epigenetic compounds with reference to their effect on DNA.

Morphological and cytogenetic studies of dominant lethality induced by mitomycin C and cyclophosphamide in female germ cells. The use of Robertsonian translocations as a 'marker system' to identify the zygote pronuclei.

Dominant lethal tests were performed on female mice injected intraperitoneally with cyclophosphamide (200 mg/kg) or with mitomycin C (0.2 or 5 mg/kg) at the preovulatory stage of oogenesis. Complementary experiments were undertaken to clarify the results obtained. Embryo culture showed that sterility found after treatment with cyclophosphamide or with the high dose of mitomycin C was the reflection of true dominant lethal effects. Mortality after cyclophosphamide treatment occurred predominantly at the 2- and 3-cell stages, while it was reported in all preimplantation stages after treatment with the high dose of mitomycin C. Embryos treated with the low dose of mitomycin C developed normally to the blastocyst stage, confirming the absence of preimplantation effects found with this dose in the dominant lethal test. Cytogenetic analysis of female pronuclei at the first cleavage division were performed after mating treated females with males homozygous for one Robertsonian translocation. This method allowed one to distinguish easily the female pronuclei from the male ones, which exhibited one translocated 'marker' chromosome. After treatment with cyclophosphamide, most female pronuclei showed multiple chromatid exchanges or shattering of the entire genome. After treatment with the high dose of mitomycin C, various types of premature chromosome condensation were found, and they were often accompanied by important interchromosome associations. After treatment with the low dose of mitomycin C, no structural chromosome aberrations were found, and the number of numerical anomalies was not significantly different from that found in control embryos. These last results suggest that the increase in rate of postimplantation loss obtained in the dominant lethal test with the low dose of mitomycin C was not due to clastogenic effects of this compound in the female germ cells, but rather to indirect effects on the maternal organism.

The effects of low-dose vincristine on megakaryocyte colony-forming cells and megakaryocyte ploidy.

The administration of low-dose vincristine (VCR) (0.1 mg/kg) to mice resulted in thrombocytosis without prior thrombocytopenia. No significant changes in marrow megakaryocyte numbers were found. However, after a minor early decrease, mean megakaryocyte ploidy increased, with a peak at 3 d. The number of megakaryocyte colony-forming cells (MEG-CFC) in bone marrow did not change significantly. In contrast with the effects on marrow, the concentration of megakaryocytes and the content of MEG-CFC in the spleen were significantly reduced for 1-2 d after VCR. This reduction was followed by a compensatory rise in the splenic content of MEG-CFC (peak 3-fold increase at 3 d), and 1-2 d later, an increase in splenic megakaryocytes which was concurrent with the increased platelet count. Culture of marrow and spleen cells in the presence of VCR resulted in inhibition of megakaryocyte colony formation at concentrations greater than 5 ng/ml and parallel reduction of the number of megakaryocytes per colony and the mean ploidy of colony megakaryocytes. The results suggest that the thrombocytosis induced by low-dose VCR does not result simply from an effect on platelets, but reflects compensatory changes in megakaryopoiesis secondary to toxic suppression of megakaryocytes and their progenitors.

Nuclear chromatin changes in hepatocytes of soman treated rats.

Cytochemical and morphometric indices of DNA metabolism and nuclear transcriptional-translational capabilities were used to monitor liver responses in Sprague-Dawley X Wistar rats subcutaneously injected with 0.9 LD50 soman, a highly toxic acetylcholinesterase-inhibiting organophosphate. Thirty minutes post-soman the following were evident: (1) increased polyploidization; (2) expansion of the nuclear envelope; and (3) elevated deoxyribonucleoprotein (DNP) complex lability to acid hydrolysis. Nuclear changes were accompanied by cytoplasmic correlates of impaired hepatocyte function, i.e., vacuolation and depressed basophilia. The overall data suggest that with acute soman toxication there are concomitant adaptive and maladaptive responses occurring within liver parenchymal elements. It is postulated that nuclear changes represent an early proliferative response and DNP activation to enhance detoxification capabilities, whereas cytoplasmic changes reflect the disruption of normal function and hepatotoxic injury.

Nocodazole and cytochalasin D induce tetraploidy in mammalian cells.

Nocodazole, a rapidly reversible inhibitor of microtubule assembly is useful for preparing mammalian cells synchronized at all stages of mitosis. When synchronized cells are allowed to progress through mitosis in the presence of cytochalasin D, the cleavage furrow is inhibited and dikaryon cells are formed. These cells become homogeneous populations of stable mononuclear tetraploid cells after the following cell division. This procedure is applicable to a wide range of mammalian cells in culture.