Genetic analyses of primary human malignant gliomas have demonstrated numerous alterations including the over-representation of chromosome 7, and normal or under-representation of chromosome 22 in primary untreated glioblastomas multiforme [18
]. Molecular cytogenetic analyses echoed the heterogeneity and complex genetics of these tumors identified by standard cytogenetics. The majority of malignant gliomas recur at or near the site of the original tumor, and it is thought that recurrent tumor arises from cells that survived the patient's treatment. Thus, cells in the recurrent tumor can be thought of as being selected for resistance in vivo
. This is further suggested by cells in the recurrent tumor that carry markers identified in the primary untreated cells, i.e. del(17)(p11.2) found in the LX and LXR cells shown in Figure . We have demonstrated that the heterogeneity present in primary tumors is markedly reduced in recurrent tumors suggesting that among the large number of genetic subpopulations, several may contain resistance and/or survival factors that permit selection and propagation. However, despite the presence of complex karyotypes with multiple abnormalities, cells selected for BCNU resistance in vitro
or in vivo
have a specific genetic characteristic – near diploid with over-representation of chromosome 7 and part, or all of chromosome 22 [2
]. While in recurrent tumors whole copies of chromosome 22 may be under-represented, FISH analysis demonstrated that numerous fragments of chromosome 22 were inserted in derivative and marker chromosomes. This was not found for other chromosomes when analyzed in a similar manner, and additional fragments of all other chromosomes were only randomly retained. To map the region of chromosome 22 that is over-represented in BCNU-resistant cells, we performed FISH analyses using pooled BAC probes. The over-represented region was further defined by use of individual BACs. This work established that this region encompassed 22q12.3–13.32.
When cells from recurrent tumor were further selected in vitro
for BCNU resistance, the percentage of cells containing the retained region of chromosome 22 increased, suggesting a role in BCNU resistance and/or cell survival for a gene or genes mapped to this region. One gene mapped to this region is the gene encoding the B-chain of platelet-derived growth factor (PDGF-B) [19
]. We have previously demonstrated that this gene is over-expressed in cells selected for resistance to BCNU and is probably involved in the autocrine growth of these cells [10
]; however, there is no evidence that expression of this gene is directly involved in chemotherapy resistance. Differential mRNA display and cDNA microarray analyses have not demonstrated consistent over-expression of additional specific genes mapped to this area in cells selected for resistance to BCNU. This suggests that sequences in this region may be contributing to a novel mechanism of resistance.
We have recently begun studies of microRNA (miRNA) expression in cells selected for resistance to BCNU. Preliminary results suggest that hsa-let-7b is over-expressed in ME cells selected for BCNU resistance and MER cells before and after selection for BCNU resistance. This miRNA is mapped to chromosome 22q13.31, the region we have found to be over-represented in BCNU resistance cells. Other miRNAs mapped to this region include hsa-let-7a-3 which is just centromeric to let-7b and hsa-mir-33 which is mapped to 22q13.2. Neither of these miRNAs are over-expressed in these cells, suggesting that this is not simply a non-specific results of over-representation. While the precise function of these miRNAs has not been elucidated, the hsa-let7 family has been implicated in the regulation of ras [20
], and reduced expression of hsa-let7 is associated with poorer survival in lung cancer [21
]. Our current studies are aimed at the identification of the role, if any, of sequences mapped to chromosome 22 in BCNU resistance.