Decreased phorbol ester receptor and protein kinase C in P388 murine leukemic cells resistant to etoposide
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Abstract:
A variant P388 murine leukemic cell resistant to 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene)-beta-D-glucopyr anoside (etoposide) (VP-16-213) was cloned. The variant P388/VP-16 cell line was 159-fold resistant to 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene)-beta-D- glucopyranoside and showed cross-resistance to vincristine (18.9-fold) and Adriamycin (522.9-fold), determined by comparing the 50% inhibitory concentrations in a 48-h growth inhibition assay. To identify the possible role of Ca2+-phospholipid-dependent protein kinase (protein kinase C) in this drug resistance, we studied the specific phorbol ester binding component and protein kinase C in the parent and drug-resistant sublines of P388 cells. The phorbol ester receptor, as expressed by the numbers of sites per cell, significantly decreased in P388/VP-16 (57.6% of control). Scatchard analysis revealed that the variant contained a single class of binding sites. However, no difference was observed in the dissociation constants (Kd), thereby suggesting much the same affinity of receptors between the two lines. Phorbol diester analogues inhibited [20-3H]phorbol-12,13-dibutyrate binding of both the variant and control cell lines, in a stereospecific manner and consistent with their binding potency. The activity of protein kinase C, which is related to the phorbol ester receptor, significantly decreased in the variant cell. The enzyme activity, particularly in the membrane fraction of P388/VP-16 cells, was remarkably decreased. These data suggest that the decrease in the specific phorbol diester receptor and protein kinase C in the variant cells might correlate with the pleiotropic drug resistance.
Insights
A drug-resistant leukemia cell line showed decreased levels of phorbol ester receptors and protein kinase C. This reduction in protein kinase C activity may contribute to the observed pleiotropic drug resistance in cancer cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Drug resistance is a major challenge in cancer chemotherapy.
- Understanding the mechanisms of multidrug resistance (MDR) is crucial for developing effective treatments.
- Protein kinase C (PKC) has been implicated in various cellular processes, including drug resistance.
Purpose of the Study:
- To investigate the role of protein kinase C (PKC) in etoposide (VP-16-213) resistant P388 murine leukemic cells.
- To examine the expression and activity of the phorbol ester receptor and PKC in drug-resistant cell lines.
Main Methods:
- Cloning of a drug-resistant P388/VP-16 cell line.
- Growth inhibition assays to determine drug resistance fold-changes.
- Scatchard analysis to assess phorbol ester receptor binding affinity and number of sites.
- Measurement of protein kinase C activity in parent and variant cell lines.
Main Results:
- The P388/VP-16 cell line exhibited significant resistance to etoposide, vincristine, and Adriamycin.
- A substantial decrease in phorbol ester receptor sites was observed in the resistant variant cells (57.6% of control).
- Protein kinase C activity, particularly in the membrane fraction, was significantly reduced in the P388/VP-16 cells.
Conclusions:
- The reduced expression of phorbol ester receptors and decreased protein kinase C activity correlate with pleiotropic drug resistance in P388/VP-16 cells.
- These findings suggest a potential role for the phorbol ester/PKC pathway in mediating multidrug resistance in leukemia.