Separate mechanisms underlying the multidrug resistant (MDR) phenotype were identified in 2 independent approaches to select tumour cells resistant to low concentrations of doxorubicin (Dox) from the sensitive T cell leukemia cell line CCRF-CEM. The CEM/A7 cell line was selected at an initial concentration of 0.005 microgram/ml of Dox and maintained at 0.07 microgram/ml. In contrast, the CEM/A5 line was selected using an initial concentration of 0.01 microgram/ml and maintained in Dox at a concentration of 0.05 microgram/ml. P-glycoprotein expression was demonstrated in the CEM/A7 line but not the CEM/A5 line. Amplification of the mdrI gene was not observed in the CEM/A7 cell line. Both cell lines showed cross-resistance to a number of structurally unrelated cytotoxic drugs including anthracyclines and etoposide (VP-16), although only the CEM/A7 line was cross resistant to Vinca alkaloids. Immunoblots of total cell lysates of the CEM/A5 line have revealed almost undetectable levels of topoisomerase II alpha and beta in this line. Cytogenetic analyses of both lines revealed numerous karyotypic abnormalities which were present in the parental cell line as well as both resistant cell lines. The CEM/A7 line also demonstrated a duplication of part of the long arm of chromosome 7 which included the region containing the mdrI gene, a finding not seen in the parental or CEM/A5 line. CEM/A5, however, demonstrated an abnormality of chromosome 7, outside the region of the mdrI gene, and it also contained a deletion of the short arm of chromosome 2. Abnormalities in this latter region of genome have been associated with non-P-glycoprotein-mediated MDR.
While loss of the Y chromosome from the karyotype of tumor cells has frequently been found in a number of human malignancies of different types, structural alterations are a much less common finding. Prompted by the high frequency of cytogenetic Y chromosome loss found in primary non-small-cell lung cancer (NSCLC), and the fact that NSCLC karyotypes usually contain marker chromosomes of unidentified origin, we have determined the Y chromosome status of 12 NSCLC samples (7 cell lines and 5 primary tumors) at a molecular level. Of the 9 cases which did not have a cytogenetically detectable Y chromosome, 4 were negative for all the Y sequences tested. The other 5, in contrast, retained some Y chromosome sequences. In 1 case (H520), only Yq heterochromatic sequences were detected, whereas in the remaining 4 (L162, L93, L125 and L71) both Yq heterochromatic sequences and Y euchromatic sequences were retained. The region of common overlap for loss of Y euchromatin was Yp distal to the Y centromere. We hypothesize that deletion of Yp sequences may play a role in tumor progression in NSCLC due to loss of a tumor-suppressor gene.
The cell line CIPL38 was derived from the pleural effusion of a patient with small cell lung cancer. The karyotype was hyperdiploid and complex with a variable number of marker chromosomes. Two of the markers had large homogeneously staining regions (hsr), which were shown to consist of amplified MYCN by in situ hybridization. One hsr bearing a marker chromosome could not be identified with G-banding, but the other was situated on a der(14). This was elucidated further with FISH analysis, which enabled the identification of sequences of chromosome i involved in a complex rearrangement with chromosome 14 and the hsr.
Summary A major form of drug resistance in tumour cells known as classical multidrug resistance (MDR) is associated with the overexpression of the mdrl gene product, the membrane protein P-glycoprotein (P-gp), which acts as an energy-dependent drug efflux pump. In this study the inheritance of P-gp expression was examined using hybrids formed after somatic cell fusion between a drug-sensitive human T-cell leukaemia cell line, CEM/CCRF, and a drug-resistant derivative, CEM/A7, which is characterised by a clonal chromosomal duplication dup(7)(ql1.23q31.2). Fourteen hybrids, chosen at random, were analysed by reverse transcriptasepolymerase chain reaction (RT-PCR) and by binding studies involving the monoclonal antibody MRK16, which recognises an external P-gp epitope. Only two hybrids were positive for both MRK16 antibody labelling and mdrl mRNA. Partial karyotypic analysis of all hybrids revealed that only the MRK16-positive hybrids contained the duplication in chromosome 7 seen in the CEM/A7 parental MDR line. Therefore, P-gp overexpression in the MRK16-positive hybrids may be linked to the inheritance of chromosome 7 from CEM/A7 and possibly associated with the chromosome 7 abnormality.
Chromosomal in situ hybridization (ISH) has extended the scope of cytogenetic analysis to nondividing cells by the use of chromosome-specific probes detected by nonisotopic techniques. This provides a rapid and sensitive method for identifying chromosomes in interphase cells, and is useful in gauging engraftment following bone marrow transplantation, particularly when the number of cells obtained is minimal. We have performed ISH using a Y-heterochromatin-specific probe to monitor patients with malignant hematological disease who have received a sex-mismatched transplant. The results have been compared with those obtained from concurrently performed standard cytogenetic analysis. Host cells were detected by interphase cytogenetics in all patients posttransplant, at times varying from 28-1,825 days, whereas routine analysis detected host cells in only 4 patients, 3 of whom were found to be in relapse. The significance of the persistence of host cells is unknown, but it does not appear to indicate impending relapse.
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