PubMed · 10969615
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L Dogliotti, C Garufi, S Iacobelli. [Chronochemotherapy].. https://doi.org/10.1177/03008916000863s111
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The human colon adenocarcinoma cell line HT29 can be adapted to 10(-7)- 10(-4) M concentrations of methotrexate (MTX). Cells adapted to 10(-4) M MTX have an enterocyte-like phenotype with DHFR gene amplification. Presently, we hypothetized that an increased expression of folate binding protein (FBP) may participate to the MTX resistance of 10(-4) MTX HT29 cells. The cDNA FBPalpha/beta-actin ratio of amplified transcripts was 4.8- and 1.5- fold higher in 10(-4) and in 10(-7) M MTX HT29 respectively, than in standard type HT29 cells. An increase of transcript level was observed when decreasing folic acid concentration. PI-PLC cleaved 7.7 times more membrane FBP in 10(-4) M than in 10(-7) M MTX and wild type HT29 cells. In contrast to 10(-7) M MTX cells, growth of 10(-4) M MTX cells was dependent on folic acid concentration and abolished at a concentration lower than 0.9 microM. In conclusion, the adaptive mechanism of HT29 cells resistant to 10(-4) M MTX is the result of the synergistic overexpression of both DHFR and FBPalpha. Overexpression of FBPalpha may be related to the enterocyte-like phenotype of the cells.
DNA polymerase beta, one of the most inaccurate DNA synthesizing enzymes, has been shown to confer genetic instability when up-regulated in cells, a situation found in several human cancers. Here, we demonstrated that enhanced activity and expression of this enzyme occur in the human ovarian tumor 2008/C13*5.25 cells, which are resistant to the antitumor agent cisplatin and hypersensitive to 6-thioguanine. We found that translesion synthesis across platinated DNA crosslinks as well as increased incorporation into DNA of 6-thioguanine took place in the 2008/C13*5.25 cells compared to the parental 2008 cells. Such features being molecular signatures of DNA polymerase beta, these findings suggest that deregulation of its expression in cancer cells may contribute to the modulation of the response to antitumor treatments and therefore to tumor progression.
Protein phosphatase 1 (PP1) plays important roles in many different aspects of cellular activities including cell cycle control. One important function of PP1 is to activate the retinoblastoma protein pRB. Here we show that pRB is one of PP1's downstream targets during apoptosis. When HL-60 cells synchronized at the G1/S boundary were treated with pro-apoptotic cytosine arabinoside (araC), PP1alpha protein increased twofold and PP1 activity about 30% within 1 h. This was followed by pRB dephosphorylation, pRB cleavage by caspases, DNA fragmentation, the appearance of cells with <2n DNA content and finally, dying and dead cells. In vitro, pRB was protected from caspase-3 digestion by prior Cdk-mediated phosphorylation, whereas PP1alpha converted phospho-pRB into an efficient substrate for caspase-3. Introduction of active PP1alpha into HL-60 cells by electroporation was sufficient to induce characteristics of apoptosis. Similarly, araC-resistant cells, normally unable to die in response to araC, initiated apoptosis when electroporated with active PP1alpha. This was also accompanied by pRB cleavage. In contrast, introduction of inhibitor-2 delayed the onset of araC-induced apoptosis, whereas concomitant introduction of PP1alpha and inhibitor-2 completely prevented PP1alpha-induced apoptosis. These results suggest that dephosphorylation of key proteins by PP1alpha may be crucial for the initiation of apoptosis and further support the concept of PP1 serving as a potential target for anti-cancer therapy.