Characterisation of Topoisomerase Iialpha Amplification and Expression in Human Lung Cancer Cell Lines

[摘要] Work has been undertaken to characterise an amplicon on chromosome 17 in a non-small cell lung carcinoma cell line, CALU-3. There are a number of different genes in this region which have been implicated in tumorigenesis. These include topoisomerase IIalpha (TOPO IIalpha), ERBB2, NM23H1 and retinoic acid receptor alpha (RARalpha) genes. Topoisomerase IIalpha is also a target for numerous anti-cancer drugs, such as VP16 and doxorubicin. Changes in the expression of topoisomerase IIalpha have been seen to have an effect on the response of cells to such topoisomerase II inhibitors, with overexpression causing sensitivity and reduced expression causing resistance to these agents. Therefore, CALU-3 is of interest as a model to study the genetic changes which have occurred around the topoisomerase IIalpha locus. Southern hybridisation was initially used to investigate which genes were within the amplicon. From this analysis it was confirmed that the TOPO IIalpha and ERBB2 genes were amplified and discovered that the RARalpha and G-CSF genes were also involved in the amplicon. Fluorescence in situ hybridisation (FISH) has subsequently been performed with CALU-3 to visualise the extent and structure of amplification for the different genes and to find any other cytogenetic changes which may have occurred to chromosome 17. New cosmid probes for TOPO IIalpha, RARalpha, NM23H1 and NF1 genes have been developed from an Imperial Cancer Research Fund Reference Library-Database chromosome 17 library, for use in FISH analysis. By using a flow-sorted whole chromosome 17 paint, seven regions of hybridisation were observed in CALU-3. None of these regions covered an entire chromosome. Five chromosome 17-specific centromere sequences were also detected in CALU-3. It has been discovered that CALU-3 carries three large regions of TOPO IIalpha and ERBB2 gene amplification, residing on three separate chromosomes. The RARalpha gene has also been found to be amplified on the same three regions, although not as highly as TOPO IIalpha or ERBB2. Counts of hybridisation signals indicate that CALU-3 has at least eight copies of RARalpha, A cosmid containing sequences thought to be in the region of the BRCA1 gene has been used and this appears to be amplified in CALU-3 at a similar level to RARalpha. Five copies of both NFl and NM23H1 genes were also found, each associated with a chromosome 17 centromere sequence. The NM23H1 gene also appears to be inverted since it is seen in close association to the centromere. Therefore, in CALU-3 there appears to have been some gross changes to chromosome 17, including numerous translocations, as well as the formation of an amplicon involving at least five genes on 17q. Expression analysis using Western blotting and immunofluorescence has confirmed that amplification of TOPO IIalpha and ERBB2 genes in CALU-3 can be correlated with the high expression of topoisomerase Ila and erbB2 proteins. No alterations were found to the expected location of either of these proteins in CALU-3, as shown by immunofluorescence, with topoisomerase IIalpha present in the nucleoplasm and erbB2 detected in the cell membrane. Row cytometry has shown that the topoisomerase IIalpha protein from CALU-3 still appears to be cell cycle regulated. The topoisomerase II enzyme in CALU-3 is also still sensitive to inhibition, as has been demonstrated by the addition of VP16 to biochemical assays of topoisomerase II activity. More specifically, the topoisomerase IIalpha isozyme is still capable of being stabilised in a drug/enzyme/DNA complex by VP16, as shown by band depletion assay. CALU-3 is very sensitive to treatment with topoisomerase II inhibitors and it seems likely that this is as a result of amplification and overexpression of the topoisomerase IIalpha gene. There is an obvious clinical interest in this relationship since topoisomerase II inhibitors are commonly used in the treatment of a variety of cancers. However, resistance to these agents is a major problem in tumour treatment. Therefore, a resistant cell line was derived by exposure to VP16, from a sensitive CALU-3 clone (clone 4) and analysed for possible mechanisms of drug resistance. A cell line was subsequently derived which was resistant to 10-5MVP16, as well as being cross-resistant to doxorubicin and vincristine. Western analysis of CALU-3/10-5M VP16 showed no difference in topoisomerase IIalpha expression when compared to the parental clone. Subsequent biochemical assays also found no difference in catalytic activity between the CALU-3 clone 4 and CALU-3/10-5M VP16. Flow cytometry was then performed to analyse P-glycoprotein expression levels in the two cell lines. No expression of this drug efflux pump was found in either parental or resistant lines. Expression of the atypical drug resistance associated protein, MRP, was then investigated using a polyclonal antiserum from Dr. Melvin Center. By Western blot analysis it appeared that CALU-3/10-5M VP16 overexpressed MRP when compared to the parental cell line. This result was confirmed by independent analysis of a Western blot with a monoclonal antibody against MRP by Dr. Susan Cole in Ontario, Canada. Therefore, it appears that MRP may play a role in the mechanism of drug resistance in CALU-3/10-5M VP16.

[发布机构] University:University of Glasgow

[关键词] Oncology, Molecular biology