Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phorbol 12-myristate 13-acetate (TPA) increases the number of colonies surviving methotrexate (MTX) exposure in a dose-dependent manner upon short incubation with Chinese hamster ovary (CHO) cells. Seventy percent of the isolated colonies showed increased copy number for the dihydrofolate reductase gene. EGTA prevents the increase in resistance triggered by TPA. Calcium ionophore A23187 and angiotensin II also increase this resistance, suggesting that calcium is involved in this process. Protein kinase C (PKC) from CHO cells is rapidly activated by TPA, A23187 and angiotensin II. PKC inhibitors, 1-(5-Isoquinolinylsulphonyl)-2-methyl-piperazine (H-7), glycyrrhetinic acid, staurosporine and calphostin C decrease the generation of resistant colonies to MTX upon incubation with TPA. However, 5 nM staurosporine on its own increases resistance to MTX while having the ability to translocate CHO PKC. In vitro, H-7, staurosporine and calphostin C inhibit PKC activity translocated by TPA incubation with CHO cells. We conclude that PKC, the activity of which is dependent on calcium and phospholipids, is part of the pathway that leads to development of increased resistance to MTX. Thus, inhibition of PKC prevents the appearance of this resistance. Our results suggest the possibility of using non-toxic PKC inhibitors as resistance modulators in MTX chemotherapy.
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PMID:Protein kinase C inhibitors reduce phorbol ester-induced resistance to methotrexate in Chinese hamster ovary cells. 764 35

4beta-Phorbol 12-myristate 13-acetate (TPA) increases the number of colonies resistant to methotrexate (MTX), mainly by amplification of the dihydrofolate reductase (dhfr) locus. We showed previously that inhibition of protein kinase C (PKC) prevents this resistance. Here, we studied the molecular changes involved in the development of TPA-mediated MTX resistance in Chinese hamster ovary (CHO) cells. TPA incubation increased the expression and activity of DHFR. Because Sp1 controls the dhfr promoter, we determined the effect of TPA on the expression of Sp1 and its binding to DNA. TPA incubation increased Sp1 binding and the levels of Sp1 protein. The latter effect was due to an increase in Sp1 mRNA. Dephosphorylation of nuclear extracts from control or TPA-treated cells reduced the binding of Sp1. Stable transfectants of PKCalpha showed increased Sp1 binding, and when treated with MTX, developed a greater number of resistant colonies than control cells. Seventy-five percent of the isolated colonies showed increased copy number for the dhfr gene. Transient expression of PKCalpha increased DHFR activity. Over-expression of Sp1 increased resistance to MTX, and inhibition of Sp1 binding by mithramycin decreased this resistance. We conclude that one mechanism by which TPA enhances MTX resistance, mainly by gene amplification, is through an increase in Sp1 expression which leads to DHFR activation.
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PMID:Sp1 involvement in the 4beta-phorbol 12-myristate 13-acetate (TPA)-mediated increase in resistance to methotrexate in Chinese hamster ovary cells. 1138 17