Alteration of STR profiles in ovarian carcinoma cells during primary culture.

PURPOSE: Cell authentication is a necessary procedure to avoid scientific data from cell culture experiments with cross-contamination or false classification. A genetic fingerprint pattern of a specimen by short tandem repeats (STR) is self-evident. Due to high amount of chromosomal rearrangements, known in epithelia ovary cancer cells and the instable STR pattern described in other tumour entities like leukaemia, this study explores the suitability of STR profiling for primary cultured epithelial ovary cancer cells. METHODS: STR profiles of epithelial ovary cancers of 16 patients were compared with corresponding blood and corresponding primary cell cultures. The primary cell cultures of epithelial ovary tumours were passaged up to 28 times. In between, cultures were cryo conserved and recultured again, two to five times per patient. RESULTS: In two cases, the STR pattern of tumour lost alleles (1/16 and 3/16) in comparison of corresponding STR-pattern from blood. In comparison to blood, cell culture of a third case, lost four alleles (4/16) accompanied with morphologic changes after 14th passage. It is equal after cryo conservation of the seventh passage from the same patient. The only changes in STR profiles we recognized are losses of alleles. Remaining STR markers allow authentication. CONCLUSIONS: Very likely, the allelic drop-outs beyond passage 14 assume complex genetic losses of heterozygosis resulting in changed growth behaviour of cells. All other STR-profiles of remaining 15 patients analysed in this study are stable over all passages and freeze-thaw processes. Thus, ovary cancer cell cultures in research should be authenticated by STR-profile in general.

Frequent translocations of 11q13.2 and 19p13.2 in ovarian cancer.

Aberrations of chromosome arm 19p in ovarian cancer were first described decades ago and have been confirmed in recent publications, which have focused on chromosome 11 as a translocation partner. Recently, genetic analysis of the ovarian cancer cell line SKOV3 revealed a rearrangement described as der(19)t(11;19)(q13.2;p13.2), which lead to a fusion protein containing parts of HOOK2 and frame shifted ACTN3 that had unknown functionality. To evaluate the frequency of these breakpoints, we used fluorescence in situ hybridization (FISH) probes flanking these genes for interphase analysis of ovarian cancer cells. We analyzed 49 primary cell cultures of ovarian cancers using FISH probes next to these breakpoints on chromosomes 11 and 19 defined in SKOV3. Co-localizations of the signals in interphase nuclei were considered to be positive fusions when the frequency was over the experimentally calculated cutoff of 24.3% (mean average value for normal ovary cells plus three times the standard deviation). Fusions between 11q13.2 and 19p13.2 were confirmed in 22 (45%) primary cell cultures of ovarian cancers. However, by PCR, the fusion originally described in SKOV3 was not detected in any of the primary cell cultures. Our results confirm other reports and show that these regions are very frequently involved in chromosomal rearrangements in ovarian cancer. Furthermore, they reveal a significant correlation (P = 0.023) of co-localized signals of 11q13.2 and 19p13.2 with low and intermediate grades in ovarian cancer.

Aurora kinase inhibitor AZD1152 has an additional effect of platinum on a sequential application at the human ovarian cancer cell line SKOV3.

PURPOSE: The treatment of ovarian tumors is carried out with platinum medicine which can lead to incompatibilities or resistances. Thus, it is of great interest to check new medicine suitability for its application. AZD1152 is an Aurora kinase inhibitor predominantly works against Aurora kinase B involved in the chromosome segregation. Cells become polyploidy and reduce the proliferation by this impairment. To investigate whether AZD1152, may play a role in the treatment of ovarian carcinoma we serving it to the cisplatinum-resistant cell line SKOV3 alone and in combination with platinum. METHODS: We look at the proliferation, the ploidy, the phases of cell cycle and the apoptosis activity of the cells. RESULTS AND CONCLUSION: We could show that the combination of both medicines in the preclinical experiment produces a working advantage.

Breakpoint characterization of the der(19)t(11;19)(q13;p13) in the ovarian cancer cell line SKOV-3.

About 20% of ovarian carcinomas show alterations of 19p13 and/or 19q13 in the form of added extra material whose origin often is from chromosome 11. Based on earlier spectral karyotype analysis of the ovarian cancer cell line SKOV-3, which shows an unbalanced translocation der(19)t(11;19), the aim of this study was to determine the precise breakpoints of that derivative chromosome. After rough delimitation of the breakpoints of microdissected derivative chromosomes by array analysis, we designed a matrix of primers spanning 11q13.2 and 19p13.2 detecting multiple amplicons on genomic and cDNA. Sequencing the amplicons, accurate localization of both breakpoints on both chromosomes was possible and we found that exon 14 of HOOK2 from chromosome 19 and exon 2 of ACTN3 from chromosome 11 were fused in the derivative chromosome. The breakpoint in the HOOK2 gene was in an intrinsic triplet of nucleic acids leading to a shift in the ACTN3 reading frame in the derivative chromosome. This frameshift alteration should give rise to an early stop codon causing a loss of function of ACTN3. Signals in two-dimensional Western blotting exactly match to calculated molecular mass and the isoelectric point of the fusion protein.