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Induction of protein kinase C in mouse melanoma cells by retinoic acid
1Department of Biochemistry Boston University School of Medicine, Massachusetts 02118.
Abstract:
Retinoic acid inhibits the proliferation of B16 mouse melanoma cells. It also eliminates the ability of these cells to grow in soft agar. These biological actions of retinoic acid have been shown to be accompanied by an increase in the amount of cyclic AMP-dependent protein kinase and an induction of a new isozyme form (RII beta). In this report we demonstrated that retinoic acid-treated B16 melanoma cells had large increases in protein kinase C activity. This increased enzyme activity was accompanied by increases in both the number of phorbol dibutyrate binding sites and the amount of immunoreactive protein kinase C. Other treatments (melanocyte-stimulating hormone, serum deprivation) which inhibited the growth of these cells did not increase protein kinase C activity. When B16 melanoma cells were treated for a prolonged time (72 h) with phorbol dibutyrate, protein kinase C activity was barely detectable. Under these conditions, melanin production was inhibited and cell growth was accelerated. When retinoic acid was added together with phorbol dibutyrate, it prevented the growth stimulatory effect of the phorbol ester and increased protein kinase C activity. However, the absolute activity of the enzyme was still below that found in control cells and very much lower than in cells treated with retinoic acid alone. Taken together with our previous findings, we propose that the increase in protein kinase C might be part of a differentiation program induced by retinoic acid.
Insights
Retinoic acid inhibits melanoma cell growth and soft agar colony formation. This effect is linked to increased protein kinase C activity, suggesting a role in cell differentiation.
Area of Science:
- Cell Biology
- Biochemistry
- Cancer Research
Background:
- Retinoic acid is known to inhibit B16 mouse melanoma cell proliferation and soft agar growth.
- Previous studies linked these effects to increased cyclic AMP-dependent protein kinase and RII beta isozyme induction.
Purpose of the Study:
- To investigate the role of protein kinase C (PKC) in retinoic acid-induced inhibition of B16 melanoma cell growth.
- To explore the relationship between PKC activity, phorbol ester binding, and cell proliferation.
Main Methods:
- Treatment of B16 melanoma cells with retinoic acid, melanocyte-stimulating hormone, serum deprivation, and phorbol dibutyrate.
- Assay of protein kinase C activity.
- Measurement of phorbol dibutyrate binding sites.
- Quantification of immunoreactive protein kinase C.
Main Results:
- Retinoic acid treatment significantly increased protein kinase C activity, phorbol dibutyrate binding, and immunoreactive PKC levels in B16 cells.
- Other growth-inhibitory treatments did not affect PKC activity.
- Prolonged phorbol dibutyrate treatment decreased PKC activity, inhibited melanin production, and accelerated cell growth.
- Retinoic acid counteracted the growth-stimulatory effect of phorbol dibutyrate and increased PKC activity, though to a lesser extent than retinoic acid alone.
Conclusions:
- The increase in protein kinase C activity induced by retinoic acid may be a key component of the differentiation program in B16 melanoma cells.
- PKC plays a significant role in mediating the anti-proliferative and differentiation effects of retinoic acid in this melanoma model.