Sp1 involvement in the 4βâphorbol 12âmyristate 13âacetate (TPA)âmediated increase in resistance to methotrexate in Chinese hamster ovary cells
2001
Noé, Véronique | Alemany, Cristina | Nicolás, Marta | Ciudad, Carlos J.
4βâPhorbol 12âmyristate 13âacetate (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 kinaseâC (PKC) prevents this resistance. Here, we studied the molecular changes involved in the development of TPAâmediated 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 Sp1Â 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 TPAâtreated cells reduced the binding of Sp1. Stable transfectants of PKCα showed increased Sp1 binding, and when treated with MTX, developed a greater number of resistant colonies than control cells. Seventyâfive percent of the isolated colonies showed increased copy number for the dhfr gene. Transient expression of PKCα increased DHFR activity. Overâexpression 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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