Involvement of the concentrative nucleoside transporter 3 and equilibrative nucleoside transporter 2 in the resistance of T-lymphoblastic cell lines to thiopurines

Mechanisms of resistance to thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) were investigated in human leukemia cell lines. We developed two 6-MP- and 6-TG-resistant cell lines from the human T-lymphoblastic cell line (MOLT-4) by prolonged exposure to these drugs. The resistant cells were highly cross resistant to 6-MP and 6-TG, and exhibited marked reduction in cellular uptake of 6-MP (70% and 80%, respectively). No significant modification of the activities of hypoxanthine-guanine phosphoribosyl transferase, thiopurine methyltransferase or inosine monophosphate dehydrogenase was observed. Real-time PCR of concentrative nucleoside transporter 3 (CNT3) and equilibrative nucleoside transporter 2 (ENT2) of resistant cells showed substantial reductions in expression of messenger RNAs. Small interfering RNA designed to silence the CNT3 and ENT2 genes down-regulated the expression of these genes in leukemia cells. These decreases were accompanied by reduction of transport of 6-MP (47% and 21%, respectively) as well as its cytocidal effect (30% and 21%, respectively). Taken together these results show that CNT3 and ENT2 play a key role in the transport of 6-MP and 6-TG by leukemia cells. From a clinical point of view determination of CNT3 and ENT2 levels in leukemia cells may be useful in predicting the efficacy of thiopurine treatment.     

Impaired Transport as a Mechanism of Resistance to Thiopurines in Human T-Lymphoblastic Leukemia Cells

In order to better understand the mechanisms of resistance to thiopurines, we studied two sublines of the MOLT4 T-lymphoblastic leukemia cell line, resistant to 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). We found that the underlying mechanism of resistance in both resistant cell lines was a markedly reduction in initial transport of 6-MP (3- and 5-fold, respectively, in 6-MP- and 6-TG-resistant cells). No significant alteration of activities of hypoxanthine-guanine phosphoribosyl transferase, thiopurine methyltransferase or inosine monophosphate dehydrogenase, the key enzymes involved in the metabolism of thiopurines was detected. We conclude that defected initial transport of thiopurines by cells may very well explain their resistance to these drugs.     

Impaired transport as a mechanism of resistance to thiopurines in human T-lymphoblastic leukemia cells

In order to better understand the mechanisms of resistance to thiopurines, we studied two sublines of the MOLT4 T-lymphoblastic leukemia cell line, resistant to 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). We found that the underlying mechanism of resistance in both resistant cell lines was a markedly reduction in initial transport of 6-MP (3- and 5-fold, respectively, in 6-MP- and 6-TG-resistant cells). No significant alteration of activities of hypoxanthine-guanine phosphoribosyl transferase, thiopurine methyltransferase or inosine monophosphate dehydrogenase, the key enzymes involved in the metabolism of thiopurines was detected. We conclude that defected initial transport of thiopurines by cells may very well explain their resistance to these drugs.     

Metabolism and mechanisms of action of 9-(tetrahydro-2-furyl)-6-mercaptopurine in Chinese hamster ovary cells

The mechanisms of action of 9-(tetrahydro-2-furyl)-6-mercaptopurine (THFMP) have been studied in Chinese hamster ovary (CHO) cells in tissue culture. THFMP is relatively unstable in physiological buffers, being facilely converted to 6-mercaptopurine (6-MP) even in the absence of cells. Consequently, THFMP undergoes metabolic conversions characteristic of 6-MP, namely formation of 6-thioIMP and incorporation into DNA as 6-thioguanine (6-TG) nucleotide. A number of purines are capable of preventing the toxicity of THFMP in wild-type cells in a manner similar to that of 6-MP. However, exogenous purines and pyrimidines did not prevent the toxicity of THFMP to cells deficient in the enzyme, hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8; HGPRTase). Cells lacking HGPRTase were 20–40-fold resistant to 6-TG and 6-MP but were only 2–4-fold resistant to THFMP. Furthermore, the time-course for killing CHO cells deficient in HGPRTase was different from that in wild-type cells containing the enzyme. There was no apparent effect of THFMP on the utilization of precursors for DNA, RNA or protein synthesis in the enzyme-deficient mutant cell line. The results suggest that THFMP is converted non-enzymatically to 6-MP and shares its mechanisms of action in wild-type cells containing HGPRTase, i.e., inhibition of de novo purine biosynthesis and incorporation into DNA as 6-TG nucleotide. However, the mechanism of action of THFMP in cells lacking HGPRTase is probably unique and is presently unknown.     

Enhancement of 6-thioguanine cytotoxic activity with methotrexate

Studies were completed to characterize the cytotoxic and biochemical interaction of methotrexate (MTX) and 6-thioguanine (6-TG). Pretreatment of L1210 leukemia cells for 3 hr with MTX substantially enhanced the cytotoxicity of 6-TG. The LD₉₀ of 6-TG in cells pretreated with 1μM MTX was 0.9pM, compared to an LD₉₀ of 800 pM when the two drugs were given concurrently and an LD₉₀ of 30 pM resulted with 6-TG alone. HPLC analysis of intracellular metabolites demonstrated an increased conversion of 6-TG to 6-TG-nucleotides in cells pretreated with MTX. A marked enhancement of 6-TG incorporation into RNA also was noted (MTX→6-TG: 350 fmol/μg RNA vs 6-TG: 98 fmol/μg RNA). However, there was a suppression of 6-TG incorporation into DNA when cells were pretreated with MTX (MTX→6-TG: 170 fmol/μg DNA vs 6-TG: 690 fmol/μg DNA). These results suggest that: 1) an enhancement of 6-TG antileukemic activity can be obtained with MTX pretreatment, and 2) the enhancement of 6-TG cytotoxicity following MTX exposure is not associated with 6-TG incorporation into DNA, but rather with incorporation of 6-TG into RNA. This drug sequence may be beneficial in the clinical treatment of leukemia.     

Mismatch repair-mediated G2/M arrest by 6-thioguanine involves the ATR-Chk1 pathway

DNA mismatch repair (MMR) deficiency in human cancers is associated with resistance to a spectrum of clinically active chemotherapy drugs, including 6-thioguanine (6-TG). We and others have shown that 6-TG-induced DNA mismatches result in a prolonged G2/M cell cycle arrest followed by apoptosis in MMR(+) human cancer cells, although the signaling pathways are not clearly understood. In this study, we found that prolonged (up to 4 days) treatment with 6-TG (3microM) resulted in a progressive phosphorylation of Chk1 and Chk2 in MMR(+) HeLa cells, correlating temporally with a drug-induced G2/M arrest. Transfection of HeLa cells with small interfering RNA (siRNA) against the ataxia telangiectasia-related (ATR) kinase or against the Chk1 kinase destroyed the G2/M checkpoint and enhanced the apoptosis following 6-TG treatment. On the other hand, the induction of a G2/M population by 6-TG was similar in ATM(-/-) and ATM(+) human fibroblasts, suggesting that the ATM-Chk2 pathway does not play a major role in this 6-TG response. Our results indicate that 6-TG DNA mismatches activate the ATR-Chk1 pathway in the MMR(+) cells, resulting in a G2/M checkpoint response     

O6-methylguanine DNA methyltransferase and p53 status predict temozolomide sensitivity in human malignant glioma cells

Temozolomide (TMZ) is a methylating agent which prolongs survival when administered during and after radiotherapy in the first-line treatment of glioblastoma and which also has significant activity in recurrent disease. O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair enzyme attributed a role in cancer cell resistance to O6-alkylating agent-based chemotherapy. Using a panel of 12 human glioma cell lines, we here defined the sensitivity to TMZ in acute cytotoxicity and clonogenic survival assays in relation to MGMT, mismatch repair and p53 status and its modulation by dexamethasone, irradiation and BCL-X(L). We found that the levels of MGMT expression were a major predictor of TMZ sensitivity in human glioma cells. MGMT activity and clonogenic survival after TMZ exposure are highly correlated (p < 0.0001, r2 = 0.92). In contrast, clonogenic survival after TMZ exposure does not correlate with the expression levels of the mismatch repair proteins mutS homologue 2, mutS homologue 6 or post-meiotic segregation increased 2. The MGMT inhibitor O6-benzylguanine sensitizes MGMT-positive glioma cells to TMZ whereas MGMT gene transfer into MGMT-negative cells confers protection. The antiapoptotic BCL-X(L) protein attenuates TMZ cytotoxicity in MGMT-negative LNT-229 but not in MGMT-positive LN-18 cells. Neither ionizing radiation (4 Gy) nor clinically relevant concentrations of dexamethasone modulate MGMT activity or TMZ sensitivity. Abrogation of p53 wild-type function strongly attenuates TMZ cytotoxicity. Conversely, p53 mimetic agents designed to stabilize the wild-type conformation of p53 sensitize glioma cells for TMZ cytotoxicity. Collectively, these results suggest that the determination of MGMT expression and p53 status will help to identify glioma patients who will or will not respond to TMZ.     

The regulatory effect of 6-TG on lncRNA–miRNA–mRNA ceRNA network in triple-negative breast cancer cell line

Breast cancer is one of the most prevalent and recurring cancer types that leads to deaths in women. Triple-negative breast cancer (TNBC) is difficult to treat due to the lack of therapeutic targets. Many studies have focused on identifying drugs for use as alternative treatments for breast cancer. Thioguanine (6-TG) exerts antitumor effects in cancer. Increasing evidence has demonstrated that competitive endogenous ribonucleic acids (ceRNAs) are involved in cancer processes. However, the mechanism by which 6-TG regulates lncRNA–miRNA–mRNAs has not been elucidated. We evaluated the antitumor effect of 6-TG in MDA-MB-231 cells and comprehensively analyzed the RNA-Seq data of MDA-MB-231 cells treated with 6-TG. Our results showed that most tumor pathways were blocked by 6-TG. The hub genes were FN1, FLNA, FLNB, VCL, GSN, MYH10, ACTN4, KDR and EREG, and they were all down-regulated after 6-TG treatment. The coexpression network consisted of 18 microRNAs (miRNAs), 9 long noncoding RNAs (lncRNAs) and 20 mRNAs. Hsa-mir-16-5p and Hsa-mir-335-5p targeted the greatest number of mRNAs in the network. These molecules could bind to PAX8-AS1 and eliminate the inhibition of target mRNA expression. We showed that PAX8-AS1 is the main lncRNA affected by 6-TG and that PAX8-AS1 regulates the hub genes in tumor pathways by competitively binding with miR-16-5p and miR-335-5p.     

Isolation and properties of lead-resistant variants of rat glioma cells

Glial cells are thought to protect neurons from heavy-metal toxicity. To gain a better understanding of mechanisms of protection against lead compounds, a number of lead-resistant C6 rat glioma cell sublines have been isolated. After 8 mo of growth in the absence of lead nitrate, three sublines still maintain their lead-resistant phenotype. None of the lead-resistant sublines are cross-resistant to Cd(II) or Ni(II), but all are cross-resistant (in varying degrees) to Hg(II), As(III), Sb(III), and Sn(II), and one is resistant to trimethyl tin. No inducible lead resistance is seen in any glioma line. One subline has been used to create cell-cell hybrids with wild-type cells. The hybrids exhibit dominance of the lead-resistant phenotype. To identify and analyze altered gene expression at the mRNA level in the lead-resistant sublines, the differential display technique was used. Numerous differences are seen between amplified fragments from wild-type and lead-resistant cells. Candidate clones are now being analyzed to confirm the differential expression and to isolate cDNAs that confer lead resistance.     

Isolation and characterisation of a full-length genomic clone encoding a plastidic glucose 6-phosphate dehydrogenase from Nicotiana tabacum

We describe here the isolation and characterisation of the first full-length genomic clone encoding a plant glucose 6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) from Nicotiana tabacum L. cv Samsun. The gene was expressed in all tissues, including roots, leaves, stems and flowers. Comparison of the gene with other known plant G6PDH cDNAs grouped this sequence with plastidic isoforms. The protein, minus a putative plastidic transit sequence, was overexpressed in Escherichia coli as a glutathione S-transferase fusion protein. The resulting protein was shown to be immunologically related to the potato plastidic G6PDH. This suggests that the sequence described here codes for a plastidic isoform. Plastidic G6PDH mRNA was induced in both roots and leaves in response to KNO₃, and the induction in roots was approximately 4 times the response seen in leaves. Sequence analysis of the 5′-untranslated region of the genomic clone indicated the presence of several NIT2 elements, which may contribute to the control of the expression of this gene. Plastidic G6PDH mRNA levels did not appear to respond to light. Received: 28 April 2000 / Accepted: 21 July 2000     

Cloning, expression and function of phosphate transporter encoded gene in Oryza sativa L.

OsPT6:1, a phosphate transporter encoding gene from the leaf samples of Oryza sativa, was identified through PCR with specifically designed primers. The phylogenetic analysis and the conserved amino acid residue site detection suggested OsPT6:1 a possible high-affinity phosphate transporter encoding gene. In situ hybridization and RT-PCR demonstrated the expression of OsPT6:1 in both roots and leaves. The peak expression signal was observed in mesophyll cells under low phosphorus (P) induction. A homologous recombination study indicated that OsPT6:1 can enhance the Pi uptake efficiency of Pichia pastoris. At the meantime, the introduction of OsPT6:1 was able to complement the Pi uptake function of yeast cells with high-affinity phosphate transporters deficient. Those results substantiated our contention that OsPT6:1 encoded a high-affinity phosphate transporter of Oryza sativa.     

Cloning, expression and function of phosphate transporter encoded gene in Oryza sativa L.

OsPT6:1, a phosphate transporter encoding gene from the leaf samples of Oryza sativa, was identified through PCR with specifically designed primers. The phylogenetic analysis and the conserved amino acid residue site detection suggested OsPT6:1 a possible high-affinity phosphate transporter encoding gene. In situ hybridization and RT-PCR demonstrated the expression of OsPT6:1 in both roots and leaves. The peak expression signal was observed in mesophyll cells under low phosphorus (P) induction. A homologous recombination study indicated that OsPT6:1 can enhance the Pi uptake efficiency of Pichia pastoris. At the meantime, the introduction of OsPT6:1 was able to complement the Pi uptake function of yeast cells with high-affinity phosphate transporters deficient. Those results substantiated our contention that OsPT6:1 encoded a high-affinity phosphate transporter of Oryza sativa. These authors contributed equally to this work.     

Aphidicolin induces 6-thioguanine resistant mutants in human diploid fibroblasts

Cytotoxic and mutagenic effects of aphidicolin (APC), an inhibitor of DNA polymerases alpha and delta, were studied in human diploid VH-10 fibroblasts. The cells were treated (2 or 4h) with APC at concentration ranges of 10-40 microM. The effect of APC on cell survival after 4 h treatment was significantly higher than after 2 h treatment. The mutagenicity of APC was investigated at the HPRT locus, and the frequency of HPRT mutants was estimated by selection in medium containing 6-thioguanine (6-TG). Treatment of fibroblast cells with 20 microM of APC for 2 or 4 h resulted approximately in 5 or 10 times increase of 6-TG resistant mutant frequencies, respectively, compared to untreated control cells.The cell cycle analyses performed during the expression time (9-12 days) have shown that after 2 and 4h treatment with APC the cells were blocked in G2 phase during the majority of the expression period, compared to control cells. Four days after the treatment, the amount of cells in G2 phase increased about two-fold (28.6-31.8% compared to 13.5% in the untreated cells). The mode of cell death during the expression time was via necrosis, rather than apoptosis, which was demonstrated by fluorescein-diacetate (FDA)-staining and terminal dUTP nick end labeling (TUNEL)-method.