Regulation of P-glycoprotein 1 and 2 gene expression and protein activity in two MCF-7/Dox cell line subclones

Summary Intrinsic low-level resistance to anti-cancer drugs is a major problem in the treatment of gastrointestinal malignancies. To address the problem presented by intrinsically resistant tumours, we have isolated two monoclonal lines from LoVo human colon adenocarcinoma cells: LoVo/C7, which is intrinsically resistant to doxorubicin (DOX); and LoVo/C5, which shows the same resistance index for DOX as the mixed parental cell population. For comparison, we have included in the study a LoVo-resistant line selected by continuous exposure to DOX and expressing a typical multidrug resistant (MDR) phenotype. In these cell lines we have studied the expression and/or activity of a number of proteins, including P-glycoprotein 170 (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), glutathione (GSH)-dependent enzymes and protein kinase C (PKC) isoforms, which have been implicated in anti-cancer drug resistance. Intracellular DOX distribution has been assessed by confocal microscopy. The results of the present study indicate that resistance in LoVo/C7 cells cannot be attributed to alterations in P-gp, LRP or GSH/GSH-dependent enzyme levels. Increased expression of MRP, accompanied by alterations in the subcellular distribution of DOX, has been observed in LoVo/C7 cells; changes in PKC isoform pattern have been detected in both intrinsically and pharmacologically resistant cells.

Section: Discussionmentioning

confidence: 71%

Characterization of a clonal human colon adenocarcinoma line intrinsically resistant to doxorubicin

Dolfini

Dasdia²,

Arancia³

et al. 1997

“…doxorubicin-. vinblastine-and colchicineresistant KB human epidermoid carcinoma cells (Davies et al 1996) and in KB-A 10 cell lines (Posada et al 1989b).…”

Section: Resultsmentioning

confidence: 99%

Inhibition of protein kinase C-α isoform enhances the P-glycoprotein expression and the survival of LoVo human colon adenocarcinoma cells to doxorubicin exposure

Porta

Dolfini²,

Comolli³

1998

Summary The aim of the present paper was to analyse the effect of long-term inhibitory treatment, for at least 7 days, of individual protein kinase C (PKC) isoforms on the survival of LoVo human colon adenocarcinoma cells to doxorubicin exposure. The treatment for 2 h, after plating the cells, and after 3 days with 1 gM Go6976, a specific inhibitor of protein kinase C (PKC)-a and -i1 isoforms, induced on day 7 in LoVo cell lines (WT) a significant increased survival when these cells were exposed to increasing doxorubicin concentrations. In contrast. resistant LoVo cells (DX) did not show significant changes in the survival to doxorubicin exposure when incubated with the inhibitor of the same specific PKC isoforms. In addition, G66976 reduced the PKC-a activity (the main calcium-dependent PKC isoforms expressed) in both cell lines with contemporary increased expression. Under such conditions. an increased nuclear activity and an increased P-glycoprotein expression occurred only in WT-treated cells with respect to untreated cells. Taken together, our data indicate a specific relationship between PKC-a inhibition, the increased nuclear PKC-a activity as well as the increased expression of P-glycoprotein. possibly causing the acquisition of a resistant phenotype in WT LoVo cells.

“…Mdr cells are characterized by an increased rate of both MDRJ transcription and gene amplification (Morrow et al, 1992;Madden et al, 1993;Davies et al, 1996). On level 20 weeks after drug removal, P-gp levels declined and, concomitantly, cells commenced to regain sensitivity towards doxorubicin.…”

Section: Discussionmentioning

confidence: 99%

“…The following two conclusions help characterize the nature of the link between mdr and PKC: (1) the decrease in mdr phenotype and the restoration of the PKC expression pattern to that observed in wild-type cells are remarkably synchronous; (2) apart from PKC-a, PKC-0 may play a role in the maintenance of the mdr phenotype. Protein levels of both these isoenzymes are elevated in MCF-7/Adr compared with wild-type MCF-7 cells, whereas those of PKC-s are decreased (Blobe et al, 1993;Davies et al, 1996). PKC phosphorylates P-gp at three serine sites within the linker region of the P-gp molecule (Chambers et al, 1995).…”

Section: Discussionmentioning

confidence: 99%

Co-ordinate loss of protein kinase C and multidrug resistance gene expression in revertant MCF-7/Adr breast carcinoma cells

Budworth

Gant²,

Gescher³

1997

Self Cite

Summary The aim of this study was to investigate the link between protein kinase C (PKC) and multidrug resistance (mdr) phenotype. The expression of both was studied in doxorubicin-resistant MCF-7/Adr cells as they reverted to the wild-type phenotype when cultured in the absence of drug. The following parameters were measured in cells 4, 10, 15, 20 and 24 weeks after removal of doxorubicin; (1) sensitivity of the cells towards doxorubicin; (2) levels of P-glycoprotein (P-gp) and MDR1 mRNA; (3) levels and cellular localization of PKC isoenzyme proteins cx, 0 and e; and (4) gene copy number of PKC-a and MDR1 genes. Cells lost their resistance gradually with time, so that by week 24 they had almost completely regained the drug sensitivity seen in wild-type MCF-7 cells. P-gp levels measured by Western blot mirrored the change in doxorubicin sensitivity. By week 20, P-gp had decreased to 18% of P-gp protein levels at the outset, and P-gp was not detectable at week 24. Similarly, MDR1 mRNA levels had disappeared by week 24. MCF-7/Adr cells expressed more PKCs-a and -0 than wild-type cells and possessed a different cellular localization of PKC-c. The expression and distribution pattern of these PKCs did not change for up to 20 weeks, but reverted back to that seen in wild-type cells by week 24. MDR1 gene amplification remained unchanged until week 20, but then was lost precipitously between weeks 20 and 24. The PKC-a gene was not amplified in MCF-7/Adr cells. The results suggest that MCF-7/Adr cells lose MDR1 gene expression and PKC activity in a co-ordinate fashion, consistent with the existence of a mechanistic link between MDR1 and certain PKC isoenzymes.