Downregulation of mdr-1 expression by 8-Cl-cAMP in multidrug resistant MCF-7 human breast cancer cells.

Abstract

8-Cl-cAMP, a site-selective analogue of cAMP, decreased mdr-1 expression in multidrug-resistant human breast cancer cells. A sixfold reduction of mdr-1 mRNA expression by 8-Cl-cAMP began within 8 h of treatment and was associated with a decrease in the synthesis of P-glycoprotein and with an increase in vinblastine accumulation. A reduction in mdr-1 expression after 8-Cl-cAMP treatment was also observed in multidrug-resistant human ovarian cancer cell lines. 8-Cl-cAMP is known to change the ratio between the two regulatory subunits, RI and RH, of protein kinase A (PKA). We observed that RIa decreased within 24 h of 8-Cl-cAMP treatment, that RIIP increased after as few as 3 h of treatment, and that PKA catalytic activity remained unchanged during 48 h of 8-Cl-cAMP treatment. The results are consistent with the hypothesis that mdr-1 expression is regulated in part by changes in PKA isoenzyme levels. Although 8-Cl-cAMP has been used to differentiate cells in other model systems, the only differentiating effect that could be detected after 8-Cl-cAMP treatment in the MCF-7TH cells was an increase in cytokeratin expression. Evidence that the reduction of mdr-1 mRNA occurred at the level of gene transcription was obtained by measuring chloramphenicol acetyltransferase (CAT) mRNA in MCF-7TH cells transfected with an mdr-1 promoter-CAT construct prior to 8-Cl-cAMP treatment. Thus, 8-Cl-cAMP is able to downregulate mdr-1 expression and suggests a new approach to reversal of drug resistance in human breast cancer. (J. Clin.

8-Cl-cAMP antagonizes mitogen-activated protein kinase activation and cell growth stimulation induced by epidermal growth factor

“…In particular, it has been suggested that PKA induced phosphorylation of Raf-1 reducing its ability to bind Ras and then blocking the ERKs signalling (Wu et al, 1993). However, 8-Cl-cAMP is not considered an activator of PKA, conversely it is rather an antagonist of PKA-I (Rohlff et al, 1993;Cho-Chung et al, 1995;Scala et al, 1995) which is the major isoenzyme expressed in KB cells (Budillon et al, unpublished observation). In addition, we have demonstrated that 8-Cl-cAMP had no effect on Raf-1 kinase activity in these cells.…”

“…PKA-I is overexpressed in tumour cells and is increased in normal cells upon exposure to mitogenic stimuli (Miller et al, 1993;Tortora et al, 1993;Cho-Chung et al, 1995), whereas PKA-II expression is typical of terminally differentiated tissues and growth-arrested cells (Schwartz and Rubin, 1985;Cho-Chung et al, 1995). 8-Cl-cAMP treatment of tumour cells reduces PKA-I activity and concomitantly increases PKA-II activity (Rohlff et al, 1993;Budillon et al, 1995;Cho-Chung et al, 1995;Scala et al, 1995). Therefore, the catalytic activity of PKA may be affected by 8-Cl-cAMP through regulation of the expression of the two isoenzymes rather than by direct activation, as previously demonstrated (Scala et al, 1995).…”

“…8-Cl-cAMP treatment of tumour cells reduces PKA-I activity and concomitantly increases PKA-II activity (Rohlff et al, 1993;Budillon et al, 1995;Cho-Chung et al, 1995;Scala et al, 1995). Therefore, the catalytic activity of PKA may be affected by 8-Cl-cAMP through regulation of the expression of the two isoenzymes rather than by direct activation, as previously demonstrated (Scala et al, 1995). The wide range of cancer cells whose growth is inhibited by 8-Cl-cAMP suggests a common mechanism of action that is likely to involve perturbation of an ubiquitous signal transduction pathway that initiates proliferation in a broad number of different cell systems, such as the mitogen-activated Ras-dependent ERKs cascade.…”

Ex vivo cytotoxic drug evaluation by DiSC assay to expedite identification of clinical targets: results with 8-chloro-cAMP

“…However 8-Cl-cAMP does not affect the doxorubicin sensitivity of MDR-expressing cell lines (Borsellino et al, 1994), although it is able to down-regulate the expression of MDR (Glazer and Rohlff, 1994;Scala et al, 1995).…”

Transcriptional regulation of MDR genes

“…Mutations between -61 and -43 resulted in a 6-fold decrease in promoter activity when the mutant MDR1 promoter construct was transiently transfected into KB-8-5 cells, and SP1 was shown to activate the MDR1 promoter when co-transfected into Drosophila cells (Cornwell, 1993a). In addition, the activity of the SP1 phosphoprotein is regulated by protein kinase A, and it has been suggested that the down-regulation of MDR1 by the PKA inhibitor 8-Cl-cAMP (Scala, 1995;Rohlff, 1995) is mediated by SP1 (Rohlff, 1998). Although initially proposed to be a critical factor for the basal transcription of MDR1 in all cells, recent evidence suggests that this requirement may be cellspecific, since mutation of this element in several human neuroblastoma cell lines had only a small effect on basal transcription (Thayer, S. and Scotto, K., unpublished).…”