Isolation and mapping of a human septin gene to a region on chromosome 17q, commonly deleted in sporadic epithelial ovarian tumors.

Allele losses from chromosome 17 are common in sporadic ovarian tumors. Previously, we reported high rates of LOH (up to 70%) from 17q25 at the marker THH59 in a bank of malignant ovarian tumors. We have extended this study to 70 tumors with 17 markers from the long arm of chromosome 17. In most cases, the data are consistent with whole chromosome loss, but we have identified a minimal region of deletion that is centered around 4 microsatellites with zero recombination at map position 106.9 cM. A P1/BAC contig across the region (approximately 200 kb) was constructed and used to determine the precise position and order of the microsatellites. The contig was shown to hybridize to 17q25 by fluorescence in situ hybridization analysis. The DNA sequence of the entire contig was determined and analyzed by BLAST searches. A 4-kb cDNA was subsequently identified with homology to the yeast, Drosophila and mammalian septin family of genes. We have designated this gene Ovarian/Breast (Ov/Br) septin. Two splice variants were demonstrated within the 200-kb contig, which differ only at exon 1. Within the contig, approximately 45% of the septin alpha transcript was identified and 38% of the septin beta transcript. The septins are a family of genes involved in cytokinesis and cell cycle control. Their known functions are consistent with the hypothesis that the human 17q25 septin gene is a candidate for the ovarian tumor suppressor gene.

Early loss of heterozygosity on 17q in ovarian cancer. The Abe Ovarian Cancer Genetics Group.

We have studied 146 ovarian tumours (94 carcinomas, 22 tumours of low malignant potential and 30 benign tumours) for evidence of allele loss on chromosome 17p and 17q sufficient to imply the proximity of a tumour-suppressor gene. We have examined two polymorphic loci (YNZ22.2 and BHP53) on 17p13 and one on chromosome 17q (17q23-qter). Loss of heterozygosity (LOH) was detected in 34/63 (54%) informative malignant tumours at YNZ22.2 and 22/47 (47%) at BHP53; on 17q, 45/64 (70%) had LOH. Allele loss was detected in a small number of benign and borderline tumours. There was a statistically significant difference between the patterns of allele loss in serous and endometrioid groups of tumours, and allele loss occurred with significantly greater frequency on 17q than on 17p. Comparison of all malignant tumours presenting with either localized (FIGO stage I/II) or widespread (FIGO stage III/IV) disease showed that, particularly on 17q, allele loss increases in the more advanced stages. The p53 tumour-suppressor gene is implicated in ovarian carcinogenesis, and our findings suggest that an important tumour-suppressor gene may be located in the region 17q23-qter. Loss of function in this gene may be responsible for the frequently observed rapid progression of serous-type adenocarcinomas to an advanced stage.

Cytochalasin B induces abnormal chromosome segregation in Friend erythroleukaemia cells.

Friend erythroleukaemia cells were made binucleate by brief exposure to cytochalasin B (CB). The binucleate cells were then allowed to grow in the absence of the CB. They produced pseudotetraploid derivatives which subsequently showed considerable variation in chromosome number. Cells with abnormal chromosome numbers failed to form clones, and disappeared from the culture. This is in marked contrast to previously reported stable pseudotetraploid cells arising in baby hamster kidney (BHK) cells after CB treatment.

Homozygosity of the short arm of chromosome 17 in cervical carcinoma.

We have determined the frequency of heterozygosity of the short arm of chromosome 17 in 20 cervical tumours using the highly polymorphic probe pYNZ22. Only 25% of the tumours were heterozygous at this locus. This is significantly lower than the level of 86% heterozygosity for this locus in the general population indicating that loss of one allele occurs in cervical cancer. Heterozygosity for a locus on the long arm of the same chromosome showed no significant difference between the tumours and the general population indicating that genetic loss was confined to the short arm of the chromosome. The analysis of premalignant lesions showed 70% of patients were heterozygous suggesting that loss of material from the short arm of chromosome 17 took place at a late stage in tumour development. This report confirms predictions made from previous karyotypic analysis and is the first indication of allele loss on the short arm of chromosome 17 in cervical cancer.

An attempt to select pseudonormal revertants of Friend erythroleukaemia cells using cytochalasin B.

Treatment of Friend erythroleukaemia cells with cytochalasin B (CB) resulted in multinucleation and loss of viability characteristic of a virus-transformed cell line. In an attempt to isolate pseudonormal revertants of this cell line mutagenized cultures were exposed to CB and surviving clones isolated. Many of these were found to be mutants resistant to the growth inhibitory effects of CB. The proportion of such mutants was reduced by simultaneous selection in CB and cytosine arabinoside. Of 699 clones examined none consistently exhibited reduced levels of multinucleation in the presence of CB. The inability of CB to select for revertants displaying a phenotype closer to normal cells is discussed.

Reversion in thymidine kinase deficient variants of mouse lymphoma P388.

The ability of 5 independently isolated thymidine kinase-deficient clones of mouse lymphoma P388 to revert has been examined. We were unable to detect spontaneous revertants in any of the 5 clones. Treatment with the hypomethylating agent 5-azacytidine induced reversion in 4 of the clones, but the frequency of revertants was very low (less than 10(-6). The response was not dose-dependent. The mutagen EMS was capable of inducing reversion in 3 of the clones with a variable level of response. The activity of thymidine kinase in 16 revertants was determined. In half of these the level of enzyme activity was considerably greater than the original P388 cell line. The high frequency loss of thymidine kinase that occurs in these cells may represent a stable inactivation of gene activity rather than an alteration in the DNA base sequence.

The thymidine-kinase locus of Friend erythroleukaemic cells. 1. Mutation rates and properties of mutants.

Spontaneous mutation at the thymidine-kinase locus in clone 707 of the Friend cell line has been examined. The rate of mutation in BrdU resistance was found to be 2.6 x 10(-6) cell-1 generation-1. The rate of reversion to HAT resistance was found to vary from 1.1 x 10(-7) to 2.85 x 10(-6) cell-1 generation-1 in 4 BrdU-resistant clones. Of 14 mutant clones assayed for thymidine-kinase activity only one had greater than 13% the activity of wild-type cells. Fluctuation analysis showed that mutations occurred spontaneously and were not induced by the selective agent. The mutation rate at the thymidine-kinase locus is several orders of magnitude greater than those reported for several other cells lines. There does not appear to be a general genetic instability in this cell line as the mutation rate at the HGPRT locus is similar to those found in other established cell lines. It is suggested that the high forward mutation rate at the thymidine-kinase locus may be due to only one functional thymidine-kinase allele being present in cells of this clone.

Killing of Friend erythroleukaemia cells by cytochalasin B is cell cycle specific.

Exposure to 2.0 micrograms/ml cytochalasin B causes loss of viability in Friend erythroleukaemia cells. This effect is only observed however in cells undergoing mitosis.

UV sensitivity in thymidine kinase deficient mouse erythroleukaemia cells.

Wild type Friend erythroleukaemia cells (clone 707) and thymidine kinase (EC. 2.7.1.2.1) deficient derivatives were examined for sensitivity to killing by ultra-violet (UV) irradiation. Deficiency of thymidine kinase leads to increased cell killing as evidenced in all of twelve thymidine kinase deficient clones examined. A revertant thymidine kinase positive clone was found to have a level of thymidine kinase activity and UV sensitivity intermediate between wild type cells and its thymidine kinase deficient parent clone. The increased cell killing in thymidine kinase deficient clones is also reflected in increased mutagenesis by UV to 6-thioguanine resistance. It is suggested that the increased mutagenesis and sensitivity is due to defective DNA repair as a result of an imbalance in deoxyribonucleoside triphosphate pools in thymidine kinase deficient cells.

Analysis of Aprt deficient mutants of Friend erythroleukaemia cells.

Cytogenetic analysis of mutants of the Friend Erythroleukaemia cell line deficient in adenine phosphoribosyl transferase (Aprt) was undertaken to ascertain whether non-disjunctional events were involved in the production of this mutation. All mutant clones examined were found to carry two copies of chromosome 8, the chromosome to which Aprt maps. Since the two homologues are distinguishable by silver staining, it was also clear that no mutants contained two copies of one homologue having lost the other. Treatment of mutants with 5-azacytidine failed to reactivate the Aprt locus.

Uncontrolled nuclear division in normal x transformed somatic cell hybrids.

Uncontrolled nuclear division (UND), as indicated by progressive multinucleation when cell division is inhibited by cytochalasin B, is a very characteristic property of transformed cells growing in vitro. In an attempt to determine the genetic basis for this transformation phenotype we have produced a series of somatic cell hybrids between different transformed and normal cells. In all hybrids there was suppression of UND but in some this was only transient. In hybrids between transformed human cells and mouse 3T3 cells, UND was only re-expressed when the human parental cell line had been transformed by SV40 virus. The results lend tentative support to the suggestion that cellular mechanisms involved in the induction and suppression of UND may not be the same in all cell lines.

Effect of cytochalasin B on somatic cell hybrids between normal and transformed cells.

Cytochalasin B (CB) prevents cytokinesis in animal cells. In normal cells nuclear division and DNA synthesis are also blocked and the cells, held in the G1 phase of the cell cycle, remain either mononucleate or binucleate. In transformed cell lines DNA synthesis and nuclear division continue and the cells become multinucleate. We have examined the response to CB in two sets of somatic cell hybrids made between cells that display multinucleation after CB treatment and cells that do not. In a cross between transformed mouse LMTK cells and normal rat embryo lung cells, very little multinucleation was observed after treatment with CB for 7 days. The ability of the LMTK cells to form clones in soft agar was also significantly reduced in these hybrids. Segregant sub-clones that re-expressed both of these transformation phenotypes were isolated. These had reduced chromosome numbers. A second cross was made between two variants of the BHK cell line, one of which displayed a high level of multinucleation in CB while the other did not. Again the hybrids showed a response similar to that of the non-multinucleating parent. From the results obtained with these two hybrids we conclude that the multinucleation induced in transformed cells by CB behaves as a recessive character in crosses with normal cells.

Azacytidine induces reversion of thymidine kinase deficiency in Friend erythroleukemia cells.

Cells of the Friend erythroleukemia cell line show a high frequency of variants which have lost thymidine kinase activity. We have exposed a thymidine kinase-deficient clone of this cell line to a series of concentrations of azacytidine and found a dose-dependent induction of thymidine kinase-positive revertants. Maximum reversion occurred with 0.9 micrograms/ml azacytidine where the frequency of revertants amongst survivors was increased by a factor of 10(6) compared to that of control cultures. Revertants were found to have varying levels of thymidine kinase activity. We conclude that DNA methylation of one or both alleles of the thymidine kinase gene is largely responsible for the instability of this gene in Friend erythroleukemia cells.

Methylation status of oestrogen receptor-alpha gene promoter sequences in human ovarian epithelial cell lines.

We have determined the methylation status of the CpG island of the oestrogen receptor alpha gene in seven human ovarian cell lines. Cell lines expressing oestrogen receptor alpha showed no evidence of hypermethylation. In three of four cell lines that produced no detectable oestrogen receptor alpha protein, hypermethylation was observed at the NotI site of the CpG island. These results indicate that aberrant hypermethylation may be responsible for a significant proportion of epithelial ovarian tumours in which oestrogen receptor alpha expression is lost.

P53 mutation does not affect prognosis in ovarian epithelial malignancies.

Mutations of the p53 tumour suppressor gene have been found in most human cancers, including ovarian epithelial malignancies. This study investigated whether the presence or absence of p53 mutation was associated with outcome following platinum-based chemotherapy in patients with ovarian cancer. DNA samples from tumour tissue and blood were obtained from 73 patients with primary tumours, 50 of whom received platinum-based adjuvant chemotherapy. Single-strand conformation polymorphism analysis and direct DNA sequencing of exons 5-8 detected mutations in 44% (32 of 73) of tumours. These were more common in late-stage (III or IV) than in early-stage disease (I or II) (p=0.03). There was no association with histological type, volume of residual disease following surgery, or initial CA125 levels. No significant association was found between p53 status and overall survival or disease-free survival following chemotherapy. Likewise, there was no correlation between p53 mutation and response to chemotherapy as defined by normalization of CA125 levels. Tumours with p53 missense mutations recurred within a significantly shorter time than those with normal p53 (p=0.04). In addition, there was a tendency for tumours with missense mutations to have a shorter disease-free survival than those with non-missense mutations, although this did not reach statistical significance (p=0.07).

Allelic imbalance of chromosome 6q in ovarian tumours.

Previous work has implicated putative tumour-suppressor (ts) genes at 6q27 and a broad region at 6p12-q23. Here we report the results of a coded, randomised study of allelic imbalance at 12 loci on 6q on 40 pairs of coded tumour-blood pairs from patients with ovarian tumours. Our results provide clear evidence for the involvement of different regions of 6q in tumours of different histological subtypes. The involvement in serous tumours of a ts gene at the distal site is confirmed. However, proximal 6q presents a complex picture, with possibly three further ts genes: one at 6q21-23.3 involved at high frequency in benign and endometrioid tumours, another at 6q14-q15, also involved in endometrioid tumours, and a third suggested by a smallest region of deletion at 6q16.3-q21, between D6S275 and D6S300, that appears to be involved in early stage tumours. These observations point the way to a statistical study of the involvement of 6q in tumours of different histological type and staging performed on larger cohorts of samples.