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[The cytogenetic characteristics of subjects with menstrual function disorders].

The frequency and the pattern of chromosomal aberrations as well as the frequency of metaphase plates with premature chromatid disjunction, polyploidy and endoreduplication were studied in lymphocyte cultures of women with disturbances of the menstrual function and in healthy women. The frequency of metaphase plates was significantly increased in persons with Turner's syndrome and reduced in the groups with amenorrhea 1 and 2 in comparison with the group of healthy women. There was no difference in chromosomal aberration pattern. Premature chromatid disjunction is characteristic of patients with disturbances of the menstrual function and healthy women. The frequency of metaphase plates with premature chromatid disjunction was elevated in groups with Turner's syndrome, amenorrhea 1 and premature sexual maturation in comparison with the control.

Adult↗
PubMed1994

The phenotype of a "Cdc2 kinase target site-deficient" mutant of oncoprotein 18 reveals a role of this protein in cell cycle control.

Oncoprotein 18 (Op18) is a cytosolic protein that is expressed in all proliferating cells. This phosphoprotein is up-regulated in a variety of human neoplasm, and phosphorylation of its two Cdc2 kinase target sites, Ser-25 and Ser-38, fluctuates dramatically during the cell cycle. We have investigated the potential role of the Cdc2 kinase-mediated phosphorylation of these two sites by expressing a "Cdc2 kinase target site-deficient" mutant of Op18 (Op18-S25,38A), and analyzed the phenotype on the level of cell cycle regulation. The result shows that induced expression of Op18-S25,38A results in rapid accumulation of cells in the G2 phase of the cell cycle. The block in G2 seems transient, since prolonged incubation was found to result in a large fraction of the transfected cells entering S phase in the absence of mitosis, i.e. endoreduplication. In addition, a fraction (30%) of the transfected cells was blocked in mitosis. Whereas the morphology of the G2 arrested cells appeared normal, expression of Op18-S25,38A caused a serious defect during mitotic chromosome segregation. Analyses of the mechanism behind the phenotype of Op18-S25,38A suggest an essential role for Op18 during cell division and that the mutant interferes with the function of the endogenous gene product.

Amino Acid Sequence↗
PubMed1994

Tamoxifen induces hepatic aneuploidy and mitotic spindle disruption after a single in vivo administration to female Sprague-Dawley rats.

Tamoxifen has found extensive use in the treatment of all stages of human breast cancer. The efficacy of tamoxifen treatment for the prevention of second primary tumors and its chemosuppressive action in animal models have led to initiation of clinical trials to test its efficacy for prevention of this disease in women. Recently, tamoxifen has been shown to induce hepatocellular carcinomas in rats. For determination of the mechanism of induction of these tumors and assessment of the possibility of risk of human cancer development from tamoxifen treatment, female Sprague-Dawley rats (five rats per treatment) were administered tamoxifen at doses ranging from 0.3 to 35 mg/kg. One day after treatment, the rats were sacrificed, and the hepatocytes were isolated and cultured for 50 h. Colcemid was added 3 h prior to harvest, and the hepatocytes were then prepared for karyotypic evaluation. One hundred metaphase spreads were examined per animal. Tamoxifen treatment resulted in the induction of aneuploidy in approximately 70% of the examined hepatocytes at the doses used. In addition, premature condensation (2-10%) and endoreduplication (5-10%) were observed in hepatocytes of rats treated with tamoxifen. Furthermore, exchanges between chromosomes as well as chromosome breakage were observed. Examination of the cultured hepatocytes from rats treated with tamoxifen by electron microscopy demonstrated both unipolar spindles and incompletely elongated spindles. Exposure of rats to a single in vivo dose of tamoxifen produced multiple changes in rat hepatocytes including clastogenic damage at doses comparable to that administered to humans. The occurrence of aneuploidy induction, premature condensation, chromosome breakage, and improper mitotic spindle formation indicates that risk versus benefit of tamoxifen treatment should be carefully evaluated.

Aneuploidy↗
PubMed1993

[Application of DNA fingerprinting to obstetrics and gynecology].

Restriction fragment length polymorphisms (RFLP) are variations in the size of restriction fragments of genomic DNA that hybridize to specific probes. They are the consequence of changes in primary DNA sequences, most of which result from small-scale changes in DNA. Recently minisatellite DNA probes that detect many regions of great variability within the human genome have been described. Minisatellite probes consist of multiple repeated copies of a common 10-15 base pair core sequence. On hybridization to restriction enzyme digests of human DNA, they simultaneously detect many highly polymorphic minisatellites at different loci in the genome, and produce band patterns that are individual specific. The band patterns are called "DNA fingerprints" or "DNA barcode" which can be used for individual identification on forensic and legal medicine. In addition to forensic and legal medicine, DNA fingerprinting can be used in both basic research and clinical examination of obstetrics and gynecology. The RFLP bands in DNA fingerprinting are inherited as single Mendelian co-dominants and we can use such minisatellite DNA probe for the determination of zygosity in multiple pregnancy. This probe can be used for the determination of androgenesis as a cause of complete hydatidiform mole. Each polymorphic band in molar tissues could be identified as being of paternal but not maternal origin. Some polymorphic bands of paternal origin were not observed in molar tissues, indicating that endoreduplication of a normal haploid sperm or fertilization by dispermy to an anuclear oocyte with no effective genome could be the cause of complete hydatidiform mole (androgenesis).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Stabilised p53 facilitates aneuploid clonal divergence in colorectal cancer.

Mutations in the p53 tumour suppressor gene are amongst the most frequent genetic abnormalities acquired in tumours. Recent studies in vitro suggest that mutant p53 destabilises the genome and facilitates development of aneuploidy. Here, in a study of 83 colorectal carcinomas, we demonstrate that alterations in p53 (detected by immunocytochemical stabilisation) precede and apparently facilitate divergence of aneuploid sub-clones. Aneuploidy in these tumours (but not those with normal p53) is predominantly in the subtetraploid range, suggesting that endoreduplication is important in its origin. This association with a specific phase of carcinoma progression is not shared by other commonly acquired genetic abnormalities in these tumours. These observations highlight the critical role of p53 in the regulation of abnormal chromosome replication and afford an explanation for the association between p53 abnormalities, aneuploidy and biological aggression in cancer.

Adenoma↗
PubMed1996

Regulation of platelet production and function by megakaryocyte growth and development factor in nonhuman primates.

The primary physiologic regulator of platelet production, Mpl ligand, has recently been cloned and characterized. To define the regulatory role of Mpl ligand on platelet production and function we measured the effects of a recombinant truncated human Mpl ligand, megakaryocyte growth and development factor (rHu-MGDF) on megakaryocytopoiesis, platelet function, and thrombogenesis in nonhuman primates. rHu-MGDF was administered to 10 baboons for 28 days while performing pharmacokinetics and repeated measurements of the following: (1) platelet count, volume, turnover, and function ex vivo and in vitro; (2) marrow megakaryocyte number, volume, and ploidy; and (3) platelet deposition and fibrin accumulation on segments of vascular graft and endarterectomized aorta in vivo. Daily subcutaneous injections of rHu-MGDF (5 microgram/kg/d) attained plasma concentrations averaging 1,300 +/- 300 pg/mL 2 hours after injection with trough levels of 300 +/- 65 pg/mL before the next dose. These levels of rHu-MGDF incrementally increased the peripheral platelet concentration threefold by day 7 and fivefold by day 28 (P < 10(-4)) associated with a reciprocal decrease of 25% in mean platelet volumes (P < 10(-3)). Platelet mass turnover, a steady-state measure of platelet production, increased fivefold (P < 10(-4)). Platelet morphology, life span, and recovery were normal. No significant change occurred in peripheral leukocyte, neutrophil, or erythrocyte counts (P > .1 in all cases). The platelet count gradually returned to baseline within 2 weeks after discontinuing rHu-MGDF infections. Marrow megakaryocyte volume doubled (P < 10(-3)) three days after initiating rHu-MGDF therapy and the modal ploidy shifted from 16N to 64N (P < 10(-4)). Marrow megakaryocyte number increased twofold by day 7, and nearly fourfold by day 28 (P < 10(-4)), resulting in a 6.5-fold increase in marrow megakaryocyte mass (P < 10(-3)). The effects of rHu-MGDF on thrombosis were determined by comparing baseline, day 5, and day 28 rHu-MGDF-treatment measurements of 111In-platelet deposition and 125I-fibrin accumulation on segments of homologous endarterectomized aorta (EA) and vascular graft (VG) interposed in arteriovenous femoral shunts. rHu-MGDF increased 111In-platelet deposition in direct proportion to the circulating concentration of platelets for both EA and VG (r=.98 in both cases), without significant changes in fibrin accumulation (P > .5 in both cases). During the first week of rHu-MGDF treatment ex vivo platelet aggregatory responsiveness was enhanced to physiologic agonists (adenosine diphosphate, collagen, and thrombin receptor agonist peptide, TRAP1-6) (P < .05 in all cases). Although in vitro platelet aggregation was not induced by any concentration of rHu-MGDF tested (P > .5), rHu-MGDF enhanced aggregatory responses to low doses of physiologic agonists, effects that were maximal at 10 ng/mL for baboon platelets and 100 ng/mL for human platelets, and were blocked by excess soluble c-Mpl receptor. Flow cytometric expression of platelet activation epitopes was not increased on resting platelets (ligand-induced binding sites, P-selectin, or Annexin V binding sites; P > .1 in all cases). Megakaryocyte growth and development factor regulates platelet production and function by stimulating endoreduplication and megakaryocyte formation from marrow progenitor cells, and transiently enhancing platelet functional responses ex vivo. rHu-MGDF has the potential for achieving platelet hemostatic protection with minimal thrombo-occlusive risk.

Animals↗
PubMed1996

Effects of recombinant human interleukin-11 (Neumega rhIL-11 growth factor) on megakaryocytopoiesis in human bone marrow.

We examined the effects of recombinant human interleukin 11 (rhIL-11) on in vivo human hematopoiesis. Twelve women with advanced breast cancer and no evidence of bone marrow (BM) involvement were treated with 10, 25, 50, or 75 micrograms/kg/day of rhIL-11 administered subcutaneously for 14 consecutive days. Examination of bone marrow trephine biopsies obtained before and after rhIL-11 treatment revealed unchanged BM cellularity at all doses, and a statistically significant increase in megakaryocyte (MK) frequencies (from 0.5 +/- 0.1% to 1.0 +/- 0.3%) following administration of the two highest doses (p < 0.001). The BM biopsies also showed an increased proportion of immature myeloid and erythroid precursors following 14 days of treatment in all cases. The mean proportion of marrow cells stained with PC10, a monoclonal antibody (mAb) that recognizes the proliferating cell nuclear antigen (PCNA), increased from 16.3 +/- 5.7% to 45.8 +/- 17.1% (p < 0.001) following the two highest treatment doses. Most of the PC10+ cells were promyelocytes and proerythroblasts. In this same group, the proportion of PC10+ MKs increased from 28.3 +/- 11.5% to 56.8 +/- 24.3% (p < 0.01) after treatment, while megakaryocyte ploidy analysis revealed a greater number of higher ploidy (64N) megakaryocytes following rhIL-11 treatment (p < 0.012). Numbers of BM and peripheral blood (PB) CD34+, CD34+DR+, and CD34+DR- cells did not change following rhIL-11 treatment. Following rhIL-11 therapy at the highest dose studied, a 3- and 10-fold increase in the number of committed BM MK progenitor cells (CFU-MK) was observed in two of three patients, while no change was seen in the number of the other BM or PB progenitor cells examined. rhIL-11 administration was also associated with an increase in BM reticulin content (fibrosis grade 1-2) in 7 patients. These results indicate that the administration of rhIL-11 to patients with normal hematopoiesis stimulates MK endoreduplication, PCNA expression, and, at high doses, increases MK and CFU-MK progenitor cell numbers. In addition, rhIL-11 was able to stimulate precursor cells of different marrow lineages without affecting the number of assayable progenitor cells.

Administration, Cutaneous↗
PubMed1996

FLT3 receptor expression on the surface of normal and malignant human hematopoietic cells.

FLT3 ligand is a hematopoietic growth factor that plays a key role in growth of primitive hematopoietic cells. FLT3 receptor mRNA is found in early hematopoietic progenitors and in human myeloid leukemia blasts. Much less is known about the surface expression of FLT3 receptor on human hematopoietic cells. Using human 125I-FLT3 ligand, we have identified and characterized surface FLT3 receptors on normal and malignant human hematopoietic cells and cell lines. Our results showed that surface display of FLT3 receptor was greatest in fresh myeloid leukemia blast cells and myeloid leukemia cell lines. Erythroleukemic and megakaryocytic leukemia cell lines (n = 5) bound little to no 125I-FLT3 ligand. Scatchard analysis of 125I-FLT3 ligand binding data shows that three myeloid leukemia cell lines, ML-1, AML-193, and HL-60, as well as normal human marrow mononuclear cells, exhibit high affinity FLT3 receptors. Crosslinking of 125I-FLT3 ligand to FLT3 receptors on the surface of ML-1 myeloid leukemia cells indicates that the FLT3 ligand. The rates of FLT3 ligand internalization and degradation were determined by binding 125I-FLT3 ligand to ML-1 cells and acid stripping to distinguish surface bound from internalized ligand. Internalized 125I-FLT3 ligand was detected within 5 minutes after binding to ML-1 cells. In addition, we evaluated the effect of FLT3 ligand on megakaryocytic colony growth and nuclear endoreduplication, alone or in the presence of thrombopoietin. FLT3 ligand did not promote colony forming unit megakaryocyte (CFU-Meg) colony growth or megakaryocyte nuclear maturation, nor did FLT3 ligand augment the effects of thrombopoietin on these measures of megakaryopoiesis. These data indicate that the FLT3 receptor shares several characteristics with the c-kit receptor including dimerization and rapid internalization. However, the more restricted cellular distribution of the FLT3 receptor may target the effects of FLT3 ligand to primitive hematopoietic cells and to myeloid and lymphoid progenitor cells, in contrast to the pleiotropic effects of the c-kit receptor ligand, stem cell factor.

Autoradiography↗
PubMed1977

Human urinary bladder carcinoma cell line (T24) in long-term culture: chromosomal studies on a wild population and derived sublines.

Karyotype changes of human urinary bladder carcinoma T24 cell line were studied in the course of 5-year in vitro cultivation. It has been found that the modal number of chromosomes gradually shifted from hypotetraploidy to triploidy during long-term cultivation. The number of endoreduplications was decreasing simultaneously and minute chromosomes occurring in a number of 4--5 in early passages appeared only rarely in later passages. Using conventional Giemsa staining, persistent marker chromosomes were detected in the wild population as well as in the eight sublines derived from T24 cells by cloning. The marker chromosomes were: metacentrics, subtelocentrics, telocentrics and long acrocentrics. Occasionally, there were found dicentrics, double-minutes, breaks and pulverization. The most characteristic marker was the metacentric chromosome the length of which corresponded approximately to the arm length of the chromosome No. 1. The metacentric chromosome in a number of 1--3 was present in 100% of T24 cells of the wild population and derived sublines at all passage levels examined.

Cell Line↗
PubMed1996

[Induction of hyperdiploidy and chromosome breaks in LIM1215 cells expressing the exogenous mutant p53].

The effect of modifications of p53 expression on the incidence of numerical and structural chromosome aberrations was studied. Infection of LIM1215 cells containing two alleles of the wild-type p53 gene (P53wt) with the recombinant viruses that expressed mutant cDNAs coding for human p53 (His273, Trp248, and His175) resulted in appearance of hyperdiploid cells in populations and an increased proportion of metaphases with chromosome breakage. Expression of the exogenous p53wt or vectors HSG/neo and pPS/neo, which did not contain the p53 cDNA, did not induce numerical or structural chromosome aberrations. Treatment of cells with caffeine decreased the p53wt content and increased the proportion of metaphases with chromosome breaks; however, it did not induce hyperdiploidy in the majority of cell lines. Only in the subline that expressed the exogenous p53Trp248 did caffeine treatment increase the proportion of hyperdiploid variants, which was correlated with the hyperexpression of the product of the mutant allele. The increase in the frequency of chromosome breaks probably resulted from p53wt inactivation, whereas changes in chromosome number might be induced by some additional activities of p53 determined by mutations. Possible mechanisms for inducing heteroploidy by mutant p53 variants, including the role of endoreduplication in inducing hyper- and polyploidy, are discussed.

Cell Line↗
PubMed1997

Evaluation of MYC and chromosome 8 copy number in breast carcinoma by interphase cytogenetics.

We used fluorescence in situ hybridization (FISH) to determine MYC and chromosome 8 copy number on whole nuclear imprint preparations of 24 breast carcinomas, seven benign breast samples, and two phyllodes tumors. None of the benign tissues and neither of the phyllodes tumors demonstrated an increased copy number for MYC or chromosome 8, which was defined as greater than two signals in > 10% of nuclei. In contrast, 22 of 24 carcinomas demonstrated an increased MYC copy number. The modal numbers of MYC copies/nucleus were 0-2 in seven cases (29%), 3-5 in seven cases (29%), 6-9 in five cases (21%), and > 9 in five cases (21%). An increased chromosome 8 copy number was observed in 21 of 22 carcinomas with MYC gain, and the modal number of signals/nucleus was either identical to (n = 14; 64%) or less than (n = 8; 36%) the number of MYC copies. The number of MYC copies correlated with cellular DNA content, as determined by using flow cytometry. In peridiploid tumors (DNA index 0.9-1.2; n = 7), the MYC copy numbers/nucleus were 0-2 in five cases and 3-5 in two cases. In contrast, the modal MYC copy numbers/nucleus among the 11 hyperdiploid tumors (DNA index 1.3-1.9) were 0-2 in one case, 3-5 in four cases, 6-9 in five cases, and > 9 in one case. All three tetraploid/hypertetraploid carcinomas exhibited > 9 MYC copies/nucleus. We conclude that an increased MYC copy number, as detected by using interphase cytogenetics, is extremely frequent in human breast carcinomas. However, in most cases, MYC gene duplication is probably secondary to polysomy of chromosome 8 and/or genomic endoreduplication (i.e., DNA aneuploidy).

Aneuploidy↗
PubMed1997

Cytologic nuclear grading of fine needle cytopunctures of breast carcinoma. Comparison with histologic nuclear grading and image cytometric data.

OBJECTIVE: To evaluate nuclear grading on fine needle cytopunctures of breast carcinoma, which is of special interest when neoadjuvant chemotherapy is planned. STUDY DESIGN: In a prospective study, we compared cytologic grading, based on nuclear parameters (pleomorphism and mitosis), to modified Scarff-Bloom-Richardson histologic grading in 105 primary operable breast carcinomas. The results of these two nuclear grading systems were compared to Feulgen image analysis data from the corresponding cytologic samples. RESULTS: The concordance rate between the two grading systems was 76%. Concordance between cytologic and histologic grading was observed more frequently in purely invasive carcinomas (85%) than in cases combining invasive and in situ components (56%). A highly significant relationship was observed between the two grading systems and indices of proliferative activity (S-phase fraction, proliferation index, 5c exceeding rate and endoreduplication rate), particularly in concordant grading. Furthermore, nuclear area correlated with the results of the two grading systems. CONCLUSION: Cytologic nuclear grading appeared to be a reliable tool for a large proportion of breast tumors. Despite difficulties related to tumor heterogeneity, which could be detected by careful cytologic examination, it is a useful alternative to histologic grading.

Biopsy, Needle↗
PubMed1997

[Genome multiplication mechanisms in the development of albumen gland polyploid cells of Succinea lauta (Gastropoda:Pulmonata). VI. Ultrastructural research of endomitotic structure].

This ultrastructural study of endomitosis in polyploid albumen gland cells of succineid snail. S. lauta was made to extend the previous research (Anisimov, 1997). The investigation was prosecuted on ultrathin sections by means of transmission electron microscopy. The insertion of 3H-thymidine and 3H-uridine into the nuclei and the light microscopy control of nuclear ploidy allowed to identify glands on early and intensive polyploidization stages and to ignore pseudoendomitotic terminally differentiated cells. Considering ultrastructural qualities and information about the cell population kinetics enabled us to discriminate between predifferentiation (2-4c), protodifferentiation (4-8c) and promoted differentiation (8-16c) cell stages. The endomitotic cycle was observed synchronously with the development of cytoplasmatic structures and secretion production in differentiating cells of ploidy levels 4-8-16c. According to morphological condition of the chromatin and chromosomes, G1-, S- and G2-interphase periods can be determined, such as successive stages of endomitosis (endopro-, meta-, ana-, and telophase). The following main features of endomitosis are accentuated: the normal (mitotic) chromosome cycle with total compaction in endometaphase and differential decompaction in interphase; the absence of the mitotic spindle; integrity of the nuclear envelope, nucleolus and contacts between them and chromosomes; part of chromosomes split nonsimultaneously and incompletely in the endoanaphase, or endomitosis with diplochromosomes rarely occurs. Chromosome nondisjunction is a temporary process that does not lead to a permanent chromosome endoreduplication (polyteny). It is emphasized that the normal chromosome cycle is combined with a complete block of cytoplasmic structures. Possible reasons of mitotic cycle reduction during the transition of differentiating cells to endomitotic polyploidy are discussed.

Animals↗
PubMed1997

[Chromatin structure and chromosomes on preparations of interphase nuclear derivatives after removal of nuclear envelopes. I. Method of preparation and morphology of residual nuclei in human leukocytes and intestinal epitheliocytes of Chironomus].

A method of a stage by stage dismantling of the cell nucleus by a 0.5-5 min resuspending cells in physiological media containing 0.05 and 0.5% of non-ionic detergent Triton X-100 is first described. Depending on the detergent concentration and cell incubation time a suspension was obtained consisting of either practically undamaged nuclei with removed envelopes, cytoplasm-free mitotic figures and isolated envelopes gradually spreading interphase chromatin associated with nucleoplasm proteins. The article describes a technique to make from such a suspension preparations of spread and flattened derivatives of nucleus and mitotic chromosomes to use for optical microscopic research. This method allows to reveal perichromatin components of the nucleus and mitotic chromosome to study structure of chromonemic net and nucleoli of diploid nuclei of human leucocytes and non-polytenic nuclei of chironomus epitheliocytes and the discoidal structure of polynemic (low-polytenic) chromosomes from the midgut epithelium cells of chironomus larvae. An attempt was made to use the obtained preparations of flattened unenveloped nuclei in order to distinguish between human leucocytes according to the structure of their chromonemic net and silver-stained nucleolus, and to find out the manner of impregnation of argyrophilic nucleoplasm proteins. The partially isolated chironomus polynemic chromosomes have morphological manifestations of unproportional endoreduplication, local compactization of chromomers and physical association between a nucleolus and numerous of distant chromosome loci. We failed, however, to find any homology in the band pattern of such polynemic chromosomes and polytenic chromosomes of larval salivary gland cells. Individuality of chromosomes in polynemic nuclei was not so obvious due to a developed network of intra- and interchromosomal ectopic contacts. The discussion touches upon the mechanisms of isolation of nuclear envelopes and ways to restore the original shape of the unenveloped nucleus in the used hydrodynamic system with fading detergent activity. Prospects are mapped out to use this technique in the structural studies of interphase nuclei and mitotic and polynemic chromosomes.

Animals↗
PubMed1997

Sodium arsenite disturbs mitosis and induces chromosome loss in human fibroblasts.

Arsenite, a unique human carcinogen, induces many types of cytogenetic alterations, such as sister chromatid exchanges, chromosome aberrations, and endoreduplication in a variety of in vivo and in vitro systems. Cytogenetic alterations are frequently associated with cancer development. The purpose of this study was to explore how arsenite induces cytogenetic alterations in human skin fibroblasts (HFW). The present results show that treatment of G2-enriched HFW cells with 5 microM arsenite results in significant delay of cell cycle progression, accumulation of mitotic cells, and prolongation of mitosis. Arsenite-induced G2 and mitotic delay are accompanied by accumulation of cyclin B1 and hyperphosphorylation of cdc2 and Mos proteins. In addition to mitotic delay and prolongation, arsenite treatment also induced out-of-phase centromere separation and alterations of chromosome segregation, such as the appearance of c-metaphase, ball-metaphase, and lagged chromosomes. Unlike spindle poisons, arsenite at the dose range used did not inhibit the spindle fiber formation but conceivably deranges the spindle apparatus. By analyzing the karyotype of established subclones surviving arsenite injury, 18% (8 of 44) showed one chromosome loss, whereas all 26 subclones derived from the untreated cultures were diploid. Furthermore, most arsenite-treated clones manifest prolonged life span (86 +/- 18 population doublings) as compared to those derived from the untreated cultures (44 +/- 11 population doublings). Unfortunately, none became immortal. Collectively, treatment of the G2-enriched HFW cells with arsenite can disturb the mitotic events and subsequently induce chromosome loss.

Arsenites↗
PubMed1997

Uncoupling of the order of the S and M phases: effects of staurosporine on human cell cycle kinases.

The protein kinase inhibitor staurosporine (SSP) was employed to study the involvement of kinases in human cell cycle progression. Thirty to 100 ng/ml SSP blocks entry into S phase and M phase. Lack of entry into S phase is due to impaired activity of the retinoblastoma protein kinase. The requirement for any of the SSP-sensitive kinases for cell cycle progression can be abrogated in tumour cells. Therefore, these kinases act in a checkpoint network negatively controlling the initiation of S phase, M phase and cytokinesis, rather than being inherent parts of a substrate-product chain required for the initiation of the cell cycle phases. As a consequence of the lack of certain checkpoint effectors, tumour cells may endoreduplicate or binucleate in the presence of SSP. The latter processes, as well as meiosis, are naturally occurring in specialized cell types, leading to the idea that this checkpoint network controls the order of the cell cycle phases in normal cells. A model is presented where the cell cycle is envisioned as two independently running cycles, the S and the M cycle, which are controlled by intra and intercycledependent checkpoints in human somatic cells. The model accounts for the dependency of S and M phase initiation on the successful completion of the previous M and S phase, respectively, as well as entry into a resting state.

Cell Cycle↗
PubMed1998

Identification of SV40 T-antigen mutants that alter T-antigen-induced chromosome damage in human fibroblasts.

The SV40 T antigen causes numerical (aneuploidy) and structural (aberrations) chromosome damage when expressed in human diploid fibroblasts. This chromosome damage precedes the acquisition of neoplastic traits such as anchorage independence, colony formation in reduced serum growth factors, immortalization, or tumorigenicity. Therefore, chromosome damage may be important in acquiring these traits because it could provide a mutational mechanism. To determine how the T antigen causes chromosome damage, point mutations were constructed that altered previously defined biochemical functions of the T protein. Mutant T antigen constructs were introduced into human diploid fibroblasts and selected by using G418. Clones of G418r cells that expressed mutant T antigens were expanded and scored for chromosome damage. Most of these mutant T antigens caused [corrected] levels of chromosome damage similar to those caused by [corrected] the wild-type T antigen. However, some T-antigen mutants induced fewer chromosome changes. A subset of these clones that induced less chromosome damage than wild-type T were examined further. Mutant T-antigen protein levels from this subset were quantified with flow cytometry and compared with wild-type protein expression levels. Mutations of T antigen shown previously to form less stable complexes with p53 caused less chromosome damage. A mutation in the zinc finger domain of T antigen also caused less chromosome damage. Interestingly, a mutant that caused loss of the ATPase activity of T antigen caused an increase in endoreduplicated cells. Also, a correlation was noted between cells expressing very low levels of T antigen (below detection limits when using flow cytometry) and an undamaged karyotype. This correlation indicates that there is a threshold level of T-antigen expression that induces chromosome damage and that expression levels on a per-cell basis rather than on a population basis should be considered in subsequent studies.

Amino Acid Substitution↗
PubMed1998

Platelets in thrombotic disorders: quantitative and qualitative platelet disorders predisposing to arterial thrombosis.

Endogenous thrombopoietin (TPO) stimulates platelet production in nonhuman primates by inducing dose-dependent megakaryocyte development from early marrow hematopoietic progenitors and subsequent proliferation and endoreduplication. In nonhuman primates, recombinant human TPO, nonpegylated or pegylated rHu megakaryocyte growth and development factor produce log-linear responses in peak peripheral platelet counts (or peripheral platelet mass turnover) and marrow megakaryocyte volume, ploidy, number, and mass. Mpl ligands can support normal peripheral platelet concentrations after myelosuppressive chemotherapy in baboons, and correct the thrombocytopenia in human immunodeficiency virus-infected chimpanzees. Whereas mpl ligands do not induce platelet aggregation in vitro, mpl ligands enhance aggregatory responsiveness of platelets to physiologic agonists both in vitro and transiently ex vivo after treatment with mpl ligands in nonhuman primates. However, platelet recruitment into forming thrombus is not augmented by these agents when evaluated in quantitative rabbit or baboon models of platelet-dependent thrombus formation, except for the effect of platelet concentration, per se. These findings indicate that appropriate dosing of mpl ligands prevents thrombocytopenia without increasing the risk of platelet-dependent thrombo-occlusive complications.

Animals↗