Abstract
The effect of okadaic acid (OA) on proto-oncogene protein expression of c-neu, c-myc, v-rasH, EGFR, and phosphotyrosine-containing phosphoproteins (P-Tyr) was investigated in rapidly growing (RG) normal human keratinocytes (NHK) and in SV-40 virally-transformed keratinocytes (SVK) cultured in a growth factor supplemented serum-free medium as assessed by indirect immunofluorescence microscopy. P-Tyr positively stains cell surface antigens (cytoplasm) diffusely at monopolar sites in RG NHK cultures. OA-treatment intensifies cytoplasmic P-Tyr staining at localized monopolar intercellular focal adhesion (IFA) sites with reduced cytoplasmic staining. P-Tyr expression was predominate at IFA sites with little cytoplasmic staining in RG SVK cultures. OA-treatment increased monopolar P-Tyr staining and cytoplasmic staining. OA-treatment in RG NHK cultures intensified cytoplasmic staining of c-myc and EGFR (epidermal growth factor receptor) expression. OA-treatment in RG NHK and SVK cultures intensified c-neu staining at monopolar IFA sites and intensified c-neu staining at both cytoplasmic and bipolar IFA sites in RG SVK cells. OA was especially cytotoxic for SVK cells. RA treatment decreased c-neu expression in RG NHK cultures while TPA treatment has a lesser effect on both cytoplasmic and IFA sites. RA treatment also decreased P-Tyr staining in both NHK and SVK cells. Again, TPA had a lesser inhibitory effect on P-Tyr staining pattern. RA-treatment had a similar effect on P-Tyr staining of RG cultures of a mouse fibroblast cell line. These results confirm the generality of OA, RA and TPA on the regulation of oncogene expression in both normal and malignantly transformed keratinocytes.
Highlights
Oncogenes are genes that have the potential to cause cancer, and often are mutated or overexpressed in tumor cells
We investigated the effect of tetradecanoyl phorbol 13-acetate (TPA) on oncogene expression in both normal human keratinocytes (NHK) and a tumorigenic cell line of Simian Virus 40 (SV-40) transfected human keratinocytes, and contrasted it relative to the effect of okadaic acid (OA) on EGFR, c-neu, v-rasH, c-myc and on the expression of phosphotyrosine-containing phosphoproteins [30]
OA-treated cells are smaller in size and are aggregated in to clumps with the cone-shaped unipolar cytoplasmic staining oriented in a parallel manner (b)
Summary
Oncogenes are genes that have the potential to cause cancer, and often are mutated or overexpressed in tumor cells. Normal cells often possess an analog DNA sequence or proto-oncogene that is not oncogenic unless mutated or overexpressed. They can be activated by mitogenic stimuli similar to their related oncogenes such as c-myc and c-neu, which activate other plasma membraneassociated tyrosine protein kinases [1]. The polyoma DNA virus, Simian Virus 40 (SV-40) can activate oncogene expression through its transcribed large T and middle T oncoproteins. These viral oncoproteins are complex with target proteins inhibiting tumor suppressor activities of p53 tumor-suppressor protein and the retinoblastoma protein [2]. The role of phosphotyrosine-binding domains in signal transduction and cancer has been well described [3]
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