James H. Gerlach

The doxorubicin-selected lung cancer cell line H69AR is resistant to many chemotherapeutic agents. However, like most tumor samples from individuals with this disease, it does not overexpress P-glycoprotein, a transmembrane transport protein that is dependent on adenosine triphosphate (ATP) and is associated with multidrug resistance. Complementary DNA (cDNA) clones corresponding to messenger RNAs (mRNAs) overexpressed in H69AR cells were isolated. One cDNA hybridized to an mRNA of 7.8 to 8.2 kilobases that was 100- to 200-fold more expressed in H69AR cells relative to drug-sensitive parental H69 cells. Overexpression was associated with amplification of the cognate gene located on chromosome 16 at band p13.1. Reversion to drug sensitivity was associated with loss of gene amplification and a marked decrease in mRNA expression. The mRNA encodes a member of the ATP-binding cassette transmembrane transporter superfamily.

A multidrug resistance phenotype is frequently observed in animal and human cell lines selected for in vitro resistance to a single chemotherapeutic agent. Overexpression of a highly conserved cell-surface glycoprotein (P-glycoprotein) is consistently associated with this phenotype in these mutant lines. A monoclonal antibody against P-glycoprotein was used to examine tumor samples from five patients with advanced ovarian cancer for evidence of P-glycoprotein overexpression. High levels of P-glycoprotein were detected in samples from two patients suggesting that a multidrug resistance mutation may also occur in ovarian cancer. This finding has broad implications for the understanding of nonresponse to chemotherapy in a variety of human neoplasms, and may provide a rational explanation for failure of chemotherapy in treatment of advanced ovarian cancer.

A multidrug resistant variant (H69AR) of the human small cell lung cancer cell line NCI-H69 was obtained by culturing these cells in gradually increasing doses of Adriamycin up to 0.8 microM after a total of 14 months. H69AR expresses the multidrug resistant phenotype because it is cross-resistant to anthracycline analogues including daunomycin, epirubicin, menogaril, and mitoxantrone as well as to acivicin, etoposide, gramicidin D, colchicine, and the Vinca alkaloids, vincristine and vinblastine. H69AR is also similar to other multidrug resistant cell lines in that it displays little or no cross-resistance to bleomycin, 5-fluorouracil, and carboplatin. It has a slight collateral sensitivity to 1-dehydrotestosterone and lidocaine. H69AR has increased cell-cell adhesiveness compared to H69, but a similar growth rate in vitro and tumorigenicity in nude mice. When cultured in the absence of Adriamycin, there is a 40% decrease in resistance by 35 days of culture, compared to cells in continuous culture in drug, but no further decrease in resistance up to 181 days. Monoclonal antibodies to P-glycoprotein have no detectable reactivity with H69AR cells as determined by enzyme-linked immunosorbent assay and immunoblotting techniques. Thus, unlike most multidrug resistant cell lines, H69AR does not appear to express enhanced levels of P-glycoprotein. H69AR will provide a useful model for the study of multidrug resistance in human small cell lung cancer.

Multiple myeloma is a disease with a high initial chemotherapeutic response but virtually no cures due to emergence of drug resistance. A doxorubicin-resistant human myeloma cell line (8226/Dox) has been selected from the myeloma cell line RPMI8226 by continuously exposing cells to gradually increasing doses of doxorubicin. The resistant phenotype has been retained for over 2 months despite growth in drug-free medium. The resistant subline was cross-resistant to mitoxantrone, acronycine, etoposide, and vincristine. The 8226/Dox cell line remained sensitive to melphalan but acquired collateral sensitivity to dexamethasone. Intracellular doxorubicin accumulation, as measured by [14C]doxorubicin and high-performance liquid chromatography, was decreased by 54% at 1 h for 8226/Dox compared to the sensitive line. Efflux of doxorubicin was significantly greater in the resistant subline as compared to the sensitive parent cell line. Membrane analysis using immunoblotting techniques detected increased expression of the integral membrane protein P-glycoprotein (Mr 170,000) in the resistant subline. Cytogenetic analysis of 8226/Dox revealed a 7q-anomaly not seen in the parent cell line. No double minutes or homogeneously staining regions were observed. The drug sensitivity/resistance pattern of the resistant cell line correlates well with clinical observations indicating the potential of this cell line as a model for resistance in multiple myeloma.