The mechanisms whereby cells develop resistance to the cancer chemotherapeutic agent, vincristine (VCR), are not entirely known. Our unique system will provide new information about this problem. The target cells for this study are the highly resistant (2750-fold) Chinese hamster lung cells, DC-3F/VCRd-5L, which 1) have a multi-drug resistance phenotype: 2) have decreased permeability to VCR and actinomycin D; 3) contain a long homogeneously staining region (HSR) on a metaphase chromosome such as seen in other systems known to contain amplified genes; 4) overproduce an Mr 19,000 acidic protein, V19; 5) synthesize and Mr 150,000 glycoprotein, gp150, as the predominant protein species in the plasma membrane rather than an Mr 100,000 species characteristic of control DC-3F cells; and 6) exhibit a marked diminution of tumor producing capacity and striking alteration toward normal morphology and growth compared to DC-3F. V19 or an analogous protein, has been detected in five additional resistant sublines of hamster, mouse, and human cells; all have gene amplification-associated chromosomal abnormalities. V19 is a newly described protein which is a candidate product of amplified genes and whose role in normal and drug-resistant cells we are investigating. V19 function will be studied by 1) determination of amino acid composition and sequence, 2) use of specific V19 antisera to quantitate and localize V19 in target and control cells, and 3) evaluation of its role in the protein phosphorylation change observed in resistant cells. We will also explore the evidence strongly suggesting that a mechanism for VCR resistance can be defined in terms of gene amplification, and we will further investigate our recent finding that VCR-resistance is associated with altered protein phosphorylation. Mechanisms, including gene amplification, for overproduction of proteins, such as V19 and gp150, in vincristine-resistant (or control) cells will be identified and investigated by preparation of cDNA probes for in situ hybridization studies and Northern and Southern blot analyses. Enzymological studies for further definition of the altered phosphorylation phenomenon as well as further characterization of gp150 by immunochemical techniques with gp150 antisera will be undertaken. Development of sensitive techniques, e.g. specific antibody reactions, probes to specific genes, to identify low levels of resistance to VCR either in the laboratory or in human patients treated with VCR is a major long-term goal.
Showing the most recent 10 out of 19 publications
