The effect of polyoma virus,serum factors,and dibutyryl cyclic AMP on dihydrofolate reductase synthesis,and the entry of quiescent cells into S phase

Three procedures were used to induce dihydrofolate reductase synthesis in quiescent cultures of methotrexate resistant mouse fibroblasts: (1) lytic infection with polyoma virus, (2) growth stimulation by replating cells at lower density in fresh cell culture medium, and (3) the addition of fresh medium to confluent cells. Following polyoma infection, an increase in the percentage of S-phase cells began at approximately 20 hours; dihydrofolate reductase synthesis also increased following a lag of 20 hours or more, and continued to increase throughout the late phase of lytic infection, reaching values nearly fivefold greater than that originally present in the quiescent cells. When quiescent cells received fresh medium (with or without replating), the percentage of cells in S phase began to increase by 10 hours and was accompanied by an increase in dihydrofolate reductase synthesis which reached a maximum by approximately 25 hours. These observations show that the initial entry of cells into S phase following mitogenic stimulation is associated with an induction of dihydrofolate reductase synthesis. Dibutyryl cyclic AMP blocked the stimulation of dihydrofolate reductase synthesis and the increase in the percentage of S-phase cells that resulted from the addition of fresh medium to confluent cells. When dibutyryl cyclic AMP was added at various times following the addition of fresh medium, the block in the induction of dihydrofolate reductase synthesis was correlated with a corresponding block in the increase in S-phase cells. These results suggest that dibutyryl cyclic AMP blocks cells at a point in Gl prior to either the induction of dihydrofolate reductase synthesis or the beginning of S phase. The relationship between the control of dihydrofolate reductase synthesis and entry into S phase suggests some form of coordinate control over these two parameters.     

Sequential amplification of dihydrofolate reductase and multidrug resistance genes in Chinese hamster ovary cells selected for stepwise resistance to the lipid-soluble antifolate trimetrexate

We describe the development of resistance to trimetrexate and piritrexim (BW 301U) by a stepwise selection protocol in Chinese hamster ovary cells. Selection in trimetrexate resulted in initial resistance as a result of dihydrofolate reductase gene amplification. Several trimetrexate-resistant variants that display 250-340-fold and 25-50-fold resistance to lipophilic and hydrophilic antifolates, respectively, were established. Increased antifolate resistance was associated with a prominent overexpression of dihydrofolate reductase as determined from the elevated folate reductase activity, cellular labeling with fluorescein-methotrexate, and steady-state mRNA levels as a result of a consistent dihydrofolate reductase gene amplification. However, upon subsequent incremental increases in trimetrexate, further resistance was also associated with amplification of the multidrug resistance gene. This resulted in overexpression of P-glycoprotein and a subsequent 20-50-fold collateral resistance to pleiotropic drugs such as adriamycin, actinomycin D, vinca alkaloids, etoposide, and colchicine. In contrast, initial resistance following selection with low piritrexim concentrations resulted from an unknown mechanism(s) not involving overproduction of either dihydrofolate reductase or P-glycoprotein. This piritrexim resistance was shared with trimetrexate but not with methotrexate. Upon further selection with piritrexim, resistant variants emerge with amplified dihydrofolate reductase but not with multidrug resistance genes. These variants were subsequently resistant to both hydrophilic and lipophilic folate antagonists but retained sensitivity to pleiotropic drugs. The pattern of resistance with methotrexate, trimetrexate, and piritrexim shared a common mechanism, dihydrofolate reductase gene amplification, but differed regarding the additional amplification of the multidrug resistance gene in trimetrexate-resistant cells as well as the emergence of an additional unknown mechanism(s) of resistance to lipid-soluble antifolates upon initial selection in piritrexim.     

Transient inhibition of DNA synthesis by 5-fluorodeoxyuridine leads to overexpression of dihydrofolate reductase with increased frequency of methotrexate resistance

Transient but incomplete suppression of DNA synthesis by a single exposure of an asynchronous population of cells to 5-fluoro-2'-deoxyuridine (FdUrd) increases the frequency of appearance of methotrexate (MTX)-resistant colonies. This increase was greater than 10-fold following a 6-h incubation of cells with 3 microM FdUrd prior to selection in MTX, an interval one-half the normal L1210 cell cycle time. During this period of exposure to FdUrd, DNA synthesis decreased to 25% of control rates and cells accumulated at the G1/S interface. The 6-h incubation with FdUrd resulted in greater than a 2.5-fold increase in the dihydrofolate reductase protein level in the treated cell population, which was accounted for, at least in part, by increased de novo synthesis of the enzyme as assessed by [35S]methionine labeling. This increase in dihydrofolate reductase was associated with a decrease in growth inhibition by MTX. A brief reversal (2 h) of FdUrd-induced DNA synthesis inhibition by the addition of thymidine eliminated the amplification of dihydrofolate reductase and the enhanced emergence of MTX-resistant clones. Beyond this, an analysis of clones that survive MTX selection indicates that the dihydrofolate reductase gene copy in cells spontaneously resistant to 50 nM MTX and those which resulted after the additional pretreatment with FdUrd for 6 h are comparable with a 2-4-fold amplification of enzyme in most clones. These studies demonstrate that FdUrd enhancement of dihydrofolate reductase expression can have a profound effect upon the incidence and expression of MTX resistance and that dihydrofolate reductase gene amplification may be another basis for antagonism between these agents.     

Transfection of DHFR- and DHFR+ mammalian cells using methotrexate-resistant mutants of mouse dihydrofolate reductase

Site-directed mutagenesis was used to generate mutants of mouse dihydrofolate reductase more resistant to methotrexate than the wild type enzyme. The mutant genes were used to transfect either DHFR- or DHFR+ cell lines. These mutants, as well as the wild type gene, were able to confer methotrexate resistance to DHFR- CHO cells. The number of selected colonies decreased with increased concentrations of methotrexate. The number of colonies observed at 10 microM methotrexate is correlated with the Ki(MTX) of the enzyme: the higher the Ki, the higher the number of colonies for the corresponding mutant. In contrast, the transfection of DHFR+ cells gave a few numbers of colonies not different for the wild type and the mutants.     

Effects of wild-type p53 expression on the quantity and activity of topoisomerase IIalpha and beta in various human cancer cell lines.

The p53 null HL-60 cell line was transfected with plasmids coding for either the wild-type p53 or mutant p53 gene. The stable expression of wild-type p53 resulted in a significant increase in sensitivity to the topoisomerase II poisons etoposide and doxorubicin, but not to the topoisomerase II inhibitors razoxane and ADR-529. HL-60 cells expressing wild-type p53 demonstrated 8- to 10-fold more VP-16 induced DNA breaks by the alkaline elution assay. The effect of inducible expression of wild-type p53 was also studied in the p53 null erythroblastoid cell line K562 and in the human squamous carcinoma cell line SqCC. The inducible expression of wild-type p53 in the K562 cell line resulted in a 3-fold increase in sensitivity to VP-16. The quantity of topoisomerase IIalpha was not altered by the transfection as determined by immunoblotting, while the amount of the beta isoform was increased 2.5-fold in HL-60 cells. The topo II catalytic activity present in nuclear extracts was measured as the decatenation of kinetoplast DNA, and found to be unaltered by p53 expression. Immunostaining for topoisomerase IIalpha was substantially diminished in both stable and inducible wild-type p53 expressing cells when three different antibodies were used (two polyclonal and one monoclonal). However, the addition of VP-16 resulted in a rapid appearance of nuclear fluorescence for topoisomerase IIalpha. No changes in topoisomerase IIbeta immunostaining were observed. These results suggest that an epitope for topoisomerase IIalpha is concealed in cells expressing wild-type p53 and that a complex between topoisomerase IIalpha and p53 may be disrupted by the addition of antitumor drugs.     

Identification of lysine-411 in the human reduced folate carrier as an important determinant of substrate selectivity and carrier function by systematic site-directed mutagenesis

Site-directed mutagenesis was used to characterize the functional role of lysine-411, a conserved amino acid located in putative transmembrane domain (TMD) 11 of the human reduced folate carrier (hRFC). Lysine-411 was mutagenized to arginine, glutamate, and leucine, and the mutant constructs (K411R-, K411E-, and K411L-hRFC, respectively) were transfected into hRFC-deficient K562 cells. The mutant hRFC constructs were all expressed at high levels and restored 22-36% of the methotrexate (MTX) transport level in wild-type (K43-6) hRFC transfectants. Although 5-formyl tetrahydrofolate (5-CHO-H(4)PteGlu) uptake levels for both the K411E- and K411L-hRFCs were also impaired (approximately 33% and 28%, respectively), a complete restoration of the wild-type level was observed for K411R-hRFC. While loss of MTX transport activity for the K411R-hRFC transfectant was associated with an incomplete restoration of MTX sensitivity compared to K43-6 cells, these cells were similarly sensitive to Tomudex. The K411R-hRFC transfectants showed an approximately threefold decreased growth requirement for 5-CHO-H(4)PteGlu compared to K43-6 cells. The 5-CHO-H(4)PteGlu transport stimulation observed for the wild-type carrier in chloride-free buffer was also observed for K411R-hRFC, however, this response was decreased for the K411E- and K411L-hRFCs. The preservation of low levels of transport for the K411E- and K411L-hRFCs suggest that the amino acid at position 411 does not directly participate in the binding of anionic hRFC substrates. However, a functionally important role for a basic amino acid at position 411 was, nonetheless, implied by the increased MTX transport for wild-type hRFC over the K411 mutant hRFCs, and the highly selective uptake of 5-CHO-H(4)PteGlu over MTX for K411R-hRFC.     

Loss and stabilization of amplified dihydrofolate reductase genes in mouse sarcoma S-180 cell lines.

We studied the loss and stabilization of dihydrofolate reductase genes in clones of a methotrexate-resistant murine S-180 cell line. These cells contained multiple copies of the dihydrofolate reductase gene which were associated with double minute chromosomes. The growth rate of these cells in the absence of methotrexate was inversely related to the degree of gene amplification (number of double minute chromosomes). Cells could both gain and lose genes as a result of an unequal distribution of double minute chromosomes into daughter cells at mitosis. The loss of amplified dihydrofolate reductase genes during growth in the absence of methotrexate resulted from the continual generation of cells containing lower numbers of double minute chromosomes. Because of the growth advantage of these cells, they became dominant in the population. We also studied an unstably resistant S-180 cell line (clone) that, after 3 years of continuous growth in methotrexate, generated cells containing stably amplified dihydrofolate reductase genes. These genes were present on one or more chromosomes, and they were retained in a stable state.     

Photoaffinity analogues of methotrexate as folate antagonist binding probes. 1. Photoaffinity labeling of murine L1210 dihydrofolate reductase and amino acid sequence of the binding region

N alpha-(4-Amino-4-deoxy-10-methylpteroyl)-N epsilon-(4-azido-5- [125I]iodosalicylyl)-L-lysine, a photoaffinity analogue of methotrexate, is only 2-fold less potent than methotrexate in the inhibition of murine L1210 dihydrofolate reductase. Irradiation of the enzyme in the presence of an equimolar concentration of the 125I-labeled analogue ultimately leads to an 8% incorporation of the photoprobe. A 100-fold molar excess of methotrexate essentially blocks this incorporation. Cyanogen bromide digestion of the labeled enzyme, followed by high-pressure liquid chromatography purification of the generated peptides, indicates that greater than 85% of the total radioactivity is incorporated into a single cyanogen bromide peptide. Sequence analysis revealed this peptide to be residues 53-111, with a majority of the radioactivity centered around residues 63-65 (Lys-Asn-Arg). These data demonstrate that the photoaffinity analogue specifically binds to dihydrofolate reductase and covalently modifies the enzyme following irradiation and is therefore a photolabeling agent useful for probing the inhibitor binding domain of the enzyme.     

MiR-24 Tumor Suppressor Activity Is Regulated Independent of p53 and through a Target Site Polymorphism

MicroRNAs (miRNAs) are predicted to regulate approximately 30% of all human genes; however, only a few miRNAs have been assigned their targets and specific functions. Here we demonstrate that miR-24, a ubiquitously expressed miRNA, has an anti-proliferative effect independent of p53 function. Cell lines with differential p53 status were used as a model to study the effects of miR-24 on cell proliferation, cell cycle control, gene regulation and cellular transformation. Overexpression of miR-24 in six different cell lines, independent of p53 function, inhibited cell proliferation and resulted in G2/S cell cycle arrest. MiR-24 over expression in cells with wt-p53 upregulated TP53 and p21 protein; however, in p53-null cells miR-24 still induced cell cycle arrest without the involvement of p21. We show that miR-24 regulates p53-independent cellular proliferation by regulating an S-phase enzyme, dihydrofolate reductase (DHFR) a target of the chemotherapeutic drug methotrexate (MTX). Of interest, we found that a miR-24 target site polymorphism in DHFR 3′ UTR that results in loss of miR-24-function and high DHFR levels in the cell imparts a growth advantage to immortalized cells and induces neoplastic transformation. Of clinical significance, we found that miR-24 is deregulated in human colorectal cancer tumors and a subset of tumors has reduced levels of miR-24. A novel function for miR-24 as a p53-independent cell cycle inhibitory miRNA is proposed.     

Substrate and inhibitor specificity of Mycobacterium avium dihydrofolate reductase

Dihydrofolate reductase (EC 1.5.1.3) is a key enzyme in the folate biosynthetic pathway. Information regarding key residues in the dihydrofolate-binding site of Mycobacterium avium dihydrofolate reductase is lacking. On the basis of previous information, Asp31 and Leu32 were selected as residues that are potentially important in interactions with dihydrofolate and antifolates (e.g. trimethoprim), respectively. Asp31 and Leu32 were modified by site-directed mutagenesis, giving the mutants D31A, D31E, D31Q, D31N and D31L, and L32A, L32F and L32D. Mutated proteins were expressed in Escherichia coli BL21(DE3)pLysS and purified using His-Bind resin; functionality was assessed in comparison with the recombinant wild type by a standard enzyme assay, and growth complementation and kinetic parameters were evaluated. All Asp31 substitutions affected enzyme function; D31E, D31Q and D31N reduced activity by 80-90%, and D31A and D31L by > 90%. All D31 mutants had modified kinetics, ranging from three-fold (D31N) to 283-fold (D31L) increases in K(m) for dihydrofolate, and 12-fold (D31N) to 223 077-fold (D31L) decreases in k(cat)/K(m). Of the Leu32 substitutions, only L32D caused reduced enzyme activity (67%) and kinetic differences from the wild type (seven-fold increase in K(m); 21-fold decrease in k(cat)/K(m)). Only minor variations in the K(m) for NADPH were observed for all substitutions. Whereas the L32F mutant retained similar trimethoprim affinity as the wild type, the L32A mutation resulted in a 12-fold decrease in affinity and the L32D mutation resulted in a seven-fold increase in affinity for trimethoprim. These findings support the hypotheses that Asp31 plays a functional role in binding of the substrate and Leu32 plays a functional role in binding of trimethoprim.