Translational control mediated by non-coding microRNAs (miRNAs) plays a key role in the mechanism of cellular resistance to anti-cancer drug treatment. Dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS, TS) are two of the most important targets for antifolate- and fluoropyrimidine-based chemotherapies in the past 50 years. In this study, we investigated the roles of miR-215 in the chemoresistance to DHFR inhibitor methotrexate (MTX) and TS inhibitor Tomudex (TDX). The protein levels of both DHFR and TS were suppressed by miR-215 without the alteration of the target mRNA transcript levels. Interestingly, despite the down-regulation of DHFR and TS proteins, ectopic expression of miR-215 resulted in a decreased sensitivity to MTX and TDX. Paradoxically, gene-specific small-interfering RNAs (siRNAs) against DHFR or TS had the opposite effect, increasing sensitivity to MTX and TDX. Further studies revealed that over-expression of miR-215 inhibited cell proliferation and triggered cell cycle arrest at G2 phase, and that this effect was accompanied by a p53-dependent up-regulation of p21. The inhibitory effect on cell proliferation was more pronounced in cell lines containing wild-type p53, but was not seen in cells transfected with siRNAs against DHFR or TS. Moreover, denticleless protein homolog (DTL), a cell cycle-regulated nuclear and centrosome protein, was confirmed to be one of the critical targets of miR-215, and knock-down of DTL by siRNA resulted in enhanced G2-arrest, p53 and p21 induction, and reduced cell proliferation. Additionally, cells subjected to siRNA against DTL exhibited increased chemoresistance to MTX and TDX. Endogenous miR-215 was elevated about 3-fold in CD133+HI/CD44+HI colon cancer stem cells that exhibit slow proliferating rate and chemoresistance compared to control bulk CD133+/CD44+ colon cancer cells. Taken together, our results indicate that miR-215, through the suppression of DTL expression, induces a decreased cell proliferation by causing G2-arrest, thereby leading to an increase in chemoresistance to MTX and TDX. The findings of this study suggest that miR-215 may play a significant role in the mechanism of tumor chemoresistance and it may have a unique potential as a novel biomarker candidate.

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Molecular mechanism of chemoresistance by miR-215 in osteosarcoma and colon cancer cells

Updates on the cytogenetics and molecular genetics of bone and soft tissue tumors: osteosarcoma and related tumors.

p53 and chemosensitivity

Embryonic stem (ES) cell lines were used to examine the role of p53 during in vitro differentiation. Undifferentiated ES cells express high levels of p53 exclusively in the wild-type conformation, immunoprecipitable by monoclonal antibody PAb246, and p53 was found to be functionally active as determined by its ability to bind DNA specifically and to activate transcription of target genes. Differentiation in vitro resulted in a decrease in the levels of p53 and in a shift in its conformational status to the mutant form, detectable by monoclonal antibody PAb240, with a concomitant loss of functional activity. The presence of functional p53 in the undifferentiated ES cells renders them hypersensitive to UV irradiation, whereas the differentiated cells were resistant to UV treatment. ES cells lacking p53 exhibit enhanced proliferation in both the undifferentiated and differentiated state, and apoptosis accompanying differentiation was found to be reduced. Furthermore, wild-type ES cells undergoing apoptosis expressed functional p53. Expression of the temperature-sensitive p53val135 mutant in wild-type ES cells resulted in a reduction of apoptosis accompanying differentiation when it adopted a mutant conformation at 39°C. These data demonstrate that functional inactivation of p53 allows differentiating cells to escape from apoptosis, and suggest that the conformational switch could regulate the inactivation process.

/pdf/regulation-of-es-cell-differentiation-by-functional-and-2vkpqay6rl.pdf

Regulation of ES cell differentiation by functional and conformational modulation of p53.

Involvement of p53 in cell differentiation and development.

HL60 cells, which lack the p53 gene due to a deletion, were used as an in vitro model system to study the effect of wild-type p53 gene expression on hematopoietic differentiation. We transfected HL60 cells with wild-type p53 and two mutant p53 cDNAs encoding the Val to Ala mutation at codon 143 and the Arg to Trp mutation at codon 248. Flow cytometry, growth, and cytochemical analysis for alpha-napthyl butyrate esterase activity and nitroblue tetrazolium reduction indicated that wild-type p53 but not mutant p53 induced early monocytic differentiation in the transfected HL60 cells without terminal growth arrest. The wild-type p53 transfectants did not differentiate along the granulocytic pathway, even when induced with 1.25% DMSO for 6 days; rather, these cells resembled monocytic cells, confirming that wild-type p53 transfection caused these cells to become committed to differentiate along the monocytic pathway. HL60 cells transfected with wild-type p53 were more sensitive to stress, such as growth in serum-depleted medium and exposure to a chemotherapeutic agent, etoposide.

/pdf/transfection-of-wild-type-but-not-mutant-p53-induces-early-fll2rm2lly.pdf

Transfection of wild-type but not mutant p53 induces early monocytic differentiation in HL60 cells and increases their sensitivity to stress

HL-60 cells that stably express transfected wild-type (wt) p53 were used to determine whether restoration of wt p53 increased the chemosensitivity of cells that normally lack p53 activity. The wt p53 HL-60 transfectants (SN3 cells) were more sensitive than the parental (S) cells to a number of common anticancer drugs representing various mechanisms of action, whereas HL-60 cells transfected with p53 genes mutated at codons 248 and 143 were not sensitized. The sensitization ratio due to the transfected wt p53 varied from about 2-fold for cisplatin to over 50-fold for thymidine. Cells treated with the thymidylate synthase inhibitor 5-fluoro-2'-deoxyuridine (FdUrd) were used to study changes in various p53-associated gene expressions. A higher percentage of apoptotic cells among the SN3 cells was observed than among the S cells at each concentration of FdUrd. The S cells had undetectable levels of bax and high levels of bcl-2, whereas the SN3 cells had undetectable levels of bcl-2 levels and appreciable basal levels of bax. After FdUrd treatment of SN3 cells, both p53 and bax levels increased, but the induction of bax was faster than that of p53 and paralleled the appearance of apoptotic DNA laddering. FdUrd treatment induced p21 expression and increased the G1 fraction of the SN3 cells but did not induce p21 or change the phase distribution in the S cells. FdUrd treatment also induced the expression and phosphorylation of cyclin D1 in the SN3 cells but not in the S cells. These results show that transfected wt p53 confers multidrug sensitivity to HL-60 cells by re-adjustment of the expressions of apoptosis genes and displays other properties characteristic of endogenously originated wt p53.

https://clincancerres.aacrjournals.org/content/clincanres/4/5/1315.full.pdf

Restoration of wild-type p53 activity in p53-null HL-60 cells confers multidrug sensitivity.

Previous studies have shown that the cytotoxicity of fluoropyrimidines is mediated, in large part, by inhibition of the enzyme thymidylate synthase (TS). The aim of this study was to determine whether the chemosensitivity of human cancer cells to fluoropyrimidines could be increased by decreasing TS expression with antisense oligodeoxyribonucleotides (ODNs). ODNs (18-mers) targeted at the AUG translational initiation site of TS mRNA inhibited translation in a sequence- and dose-dependent manner in a rabbit reticulocyte lysate in vitro translation system. Treatment of human colon cancer HT-29 cells with antisense ODNs decreased TS catalytic activity in the cells in a dose-dependent manner over a short period, but the longer-term effect of the TS antisense ODN treatment was actually to increase the amount of TS in the cells and to decrease their sensitivity to 5-fluoro-2'-deoxyuridine (FdUrd). However, when human nasopharyngeal cancer KB31 cells were transfected with a plasmid (pHaMAGRP) construct containing the TS antisense fragment (+ 1 to + 422) under the control of a glucose-regulated promoter, the expression of both TS protein and TS catalytic activity was decreased by nearly 30% (P = 0.014), and sensitivity of these cells to FdUrd was enhanced by approximately 8-fold (P = 0.021). No changes in the levels of expression of TS protein or FdUrd-associated cytotoxicity were observed in control, vector-transfected cells. No change was observed in the sensitivity of transfected cells toward either cisplatin or Adriamycin. These results show that the level of expression of TS in human malignant cells can be down-regulated with antisense TS RNA, and their sensitivity to fluoropyrimidines can, thereby, be increased.

/pdf/desensitization-and-sensitization-of-cells-to-1qjmqmtcsh.pdf

Desensitization and sensitization of cells to fluoropyrimidines with different antisenses directed against thymidylate synthase messenger RNA.

Our previous study demonstrated that the anticancer agent cis-diamminedichloroplatinum (II) (cis-DDP) inhibited the self-splicing activity of the Tetrahymena rRNA. The present study investigated the effects of cis-DDP on pre-mRNA splicing using a HeLa cell nuclear extract. A 2-h exposure of cis-DDP inhibited the splicing of the human B-globin pre-mRNA in a concentration-dependent manner. The concentration required for 50% inhibition of splicing (IC50) was 51 microM. Complete inhibition of spliceosome assembly occurred when the extracts were incubated with 150 microM cis-DDP. The inhibition of splicing by cis-DDP occurred at early events during spliceosome formation and to a greater extent if the extract was pre-incubated with cis-DDP in the absence of pre-mRNA. Splicing was inhibited when both pre-mRNA and cis-DDP were added simultaneously to the reaction mixture but not when cis-DDP was added 30 min after splicing was initiated with pre-mRNA. Clinically useful platinum analogs (ormaplatin, carboplatin, cis-tetraplatin and iproplatin) as well as the clinically ineffective Pt(dien)C1+, compound were tested for their ability to inhibit pre-mRNA splicing. The Pt(dien)C1+ compound, which acts in a monofunctional manner only, failed to inhibit splicing. A varying degree of splicing inhibition was observed for the other platinum analogs studied; the inhibitory activity decreased in the following order: ormaplatin > cis-tetraplatin > cis-DDP > iproplatin > carboplatin. We describe a novel mechanism that may be involved in the activity and/or toxicity of platinum agents.

Jing-Fang Ju

Papers

Transfection of wild-type but not mutant p53 induces early monocytic differentiation in HL60 cells and increases their sensitivity to stress

Restoration of wild-type p53 activity in p53-null HL-60 cells confers multidrug sensitivity.

Desensitization and sensitization of cells to fluoropyrimidines with different antisenses directed against thymidylate synthase messenger RNA.

Inhibition of pre-mRNA splicing by cisplatin and platinum analogs.