Debabrata Banerjee

Novel aspects of resistance to drugs targeted to dihydrofolate reductase and thymidylate synthase

Drug resistance is often a limiting factor in successful chemotherapy. Our laboratory has been interested in studying mechanisms of resistance to drugs that are targeted to the thymidylate biosynthesis pathway especially those that target thymidylate synthase (TS) and dihydrofolate reductase (DHFR). We have used leukemia as a model system to study resistance to methotrexate (MTX) and colorectal cancer as the model system to study 5-fluorouracil (5-FU) resistance. In leukemias, we and others have shown that transport, efflux, polyglutamylation and hydrolase activities are major determinants of MTX resistance. We have further reported that some leukemic cells have an increase in DHFR gene copy number possibly contributing to the resistant phenotype. Recently, we have begun to study in detail the molecular mechanisms that govern translational regulation of DHFR in response to MTX as an additional resistance mechanism. Studies thus far involving colorectal tumors obtained from patients have focused predominantly on the predictive value of levels of TS expression and p53 mutations in determining response to 5-FU. Although the predictive value of these two measures appears to be significant, given the variety of resistance to 5-FU observed in cell lines, it is not likely that these are the only measures predictive of response or responsible for acquired resistance to this drug. The enzyme uridine-cytidine monophosphate kinase (UMPK) is an essential and rate-limiting enzyme in 5-FU activation while dihydropyrimidine dehydrogenase (DPD) is a catabolic enzyme that inactivates 5-FU. Alterations in UMPK and DPD may therefore explain failure of 5-FU response in the absence of alterations in TS or p53. Transcription factors that regulate TS may also influence drug sensitivity. We have found that mRNA levels of the E2F family of transcription factors correlates with TS message levels and are higher in lung metastases than in liver metastases of colorectal cancers. Moreover, gene copy number of the E2F-1 gene appears to be increased in a significant number of samples obtained from metastases of colorectal cancer. We have also generated mutants of both DHFR and TS that confer resistance to MTX as well as 5-FU by random as well as site-directed mutagenesis. These mutants used alone or as fusion cDNAs of the mutants have proven to be useful in transplant studies where transfer of these mutant cDNAs to bone marrow cells have been shown to confer drug resistance to recipients. The fusion cDNAs of DHFR such as the DHFR-herpes simplex virus type 1 thymidine kinase (HSVTK) are also useful for regulation of gene expression in vivo using MTX as the small molecule regulator that can be monitored by positron emission tomography (PET) scanning or by optical imaging using a fusion construct such as DHFR-EGFP.

Increased Photosensitivity in HL60 Cells Expressing Wild-Type p53

Loss of p53 function has been correlated with decreased sensitivity to chemotherapy and radiation therapy in a variety of human tumors. Comparable analysis of p53 status with sensitivity to oxidative stress induced by pho‐todynamic therapy has not been reported. In the current study we examined photosensitivity in human promye‐locytic leukemia HL60 cells exhibiting either wild‐type p53, mutated p53 or deleted p53 expression. Experiments were performed using a purpurin, tin ethyl etiopurpurin (SnET2)‐, or a porphyrin, Photofrin (PH)‐based photo‐sensitizer. Total SnET2 accumulation was comparable in all three cell lines. Uptake of PH was highest in cells expressing wild‐type p53 but incubation conditions could be adjusted to achieve equivalent cellular PH levels during experiments that analyzed photosensitivity. Survival measurements demonstrated that HL60 cells expressing wild‐type p53 were more sensitive to PH‐ and SnET2‐mediated photosensitization, as well as to UVC irradiation, when compared to HL60 cells exhibiting deleted or mutated p53 phenotypes. A rapid apoptotic response was observed following purpurin‐ and porphyrin‐induced photosensitization in all cell lines. Results of this study indicate that photosensitivity is increased in HL60 cells expressing wild‐type p53 and that photosensitizer‐medi‐ated oxidative stress can induce apoptosis through a p53‐independent mechanism in HL60 cells.

Resistance Mechanisms to Methotrexate in Tumors

The mechanisms of intrinsic and acquired resistance to methotrexate (MTX) in human tumors are reviewed herein. In blasts from patients with acute lymphocytic leukemia, resistance mechanisms found are decreased uptake and increased dihydrofolate reductase (DHFR) activity. A major cause of intrinsic resistance to MTX in soft tissue sarcoma cells and in acute myelocytic leukemia appears to be a lack of drug retention, due mainly to low levels of polyglutamylation. A novel association between lack of the retinoblastoma protein and intrinsic MTX resistance has been found. This has been attributed to an increase in DHFR activity, due to an increased rate of transcription of this gene, stimulated by an increase in levels of free E2F, not sequestered by hypophosphorylated retinoblastoma protein.

Retroviral transduction of human dihydropyrimidine dehydrogenase cDNA confers resistance to 5-fluorouracil in murine hematopoietic progenitor cells and human CD34+-enriched peripheral blood progenitor cells.

Severe 5-fluorouracil (5-FU) toxicity has been reported among patients lacking dihydropyrimidine dehydrogenase (DPD) enzymatic activity. DPD is the principal enzyme involved in the degradation of 5-FU to 5′-6′-dihydrofluorouracil, which is further metabolized to fluoro-β-alanine. We demonstrate here that overexpression of human DPD confers resistance to 5-FU in NIH3T3 cells, mouse bone marrow cells, and in human CD34+-enriched hematopoietic progenitor cells. An SFG-based dicistronic retroviral vector containing human DPD cDNA, an internal ribosomal entry site (IRES), and the neomycin phosphotransferase (Neo) gene was constructed (SFG–DPD–IRES–Neo). Transduced NIH3T3 cells demonstrated a 2-fold (ED50) increase in resistance to a 4-hour exposure of 5-FU in comparison to nontransduced cells. Expression of DPD was confirmed by Northern and Western blot analyses, and DPD enzyme activity was detectable only in transduced cells. Infection of mouse bone marrow cells with this retroviral construct resulted in an increased number of 5-FU–resistant CFU-GM colonies, compared to mock-transduced bone marrow in both 4-hour and 12- to 14-day exposures. Infection of human CD34+-enriched cells with this construct and incubation with 5-FU (10−6 M) for 14 days also resulted in an increased number of 5-FU–resistant colonies. Retroviral transduction of human hematopoietic progenitor cells with a cDNA-expressing human DPD conferred resistance to 5-FU in NIH3T3 cells, mouse bone marrow cells, and human CD34+-enriched cells. These results encourage the use of this gene as a method to protect patients from 5-FU myelotoxicity. Cancer Gene Therapy (2001) 8, 966–973

Probing the folate-binding site of human thymidylate synthase by site-directed mutagenesis. Generation of mutants that confer resistance to raltitrexed, Thymitaq, and BW1843U89.

Human thymidylate synthase (TS) contains three highly conserved residues Ile-108, Leu-221, and Phe-225 that have been suggested to be important for cofactor and antifolate binding. To elucidate the role of these residues and generate drug-resistant human TS mutants, 14 variants with multiple substitutions of these three hydrophobic residues were created by site-directed mutagenesis and transfected into mouse TS-negative cells for complementation assays and cytotoxicity studies, and the mutant proteins expressed and characterized. The I108A mutant confers resistance to raltitrexed and Thymitaq with respective IC50 values 54- and 80-fold greater than wild-type but less resistance to BW1843U89 (6-fold). The F225W mutant displays resistance to BW1843U89 (17-fold increase in IC50 values), but no resistance to raltitrexed and Thymitaq. It also confers 8-fold resistance to fluorodeoxyuridine. Both the kinetic characterization of the altered enzymes and formation of antifolate-resistant colonies in mouse bone marrow cells that express mutant TS are in accord with the IC50 values for cytotoxicity noted above. The human TS mutants (I108A and F225W), by virtue of their desirable properties, including good catalytic function and resistance to antifolate TS inhibitors, confirm the importance of amino acid residues Ile-108 and Phe-225 in the binding of folate and its analogues. These novel mutants may be useful for gene transfer experiments to protect hematopoietic progenitor cells from the toxic effects of these drugs.

Transfection with a cDNA encoding a Ser31 or Ser35 mutant human dihydrofolate reductase into Chinese hamster ovary and mouse marrow progenitor cells confers methotrexate resistance

Chinese hamster ovary (CHO) DHFR- cells were converted into the DHFR+ phenotype when they were transfected with a mammalian expression vector carrying human dihydrofolate reductase-encoding cDNAs (DHFR) containing a Ser31 or a Ser34 mutation. Furthermore, transfection of these mutants into wild-type CHO cells resulted in resistance to high levels of methotrexate (MTX), indicating that these human variants can act as dominant selectable markers. Southern blot analysis and polymerase chain reaction amplifications confirmed that the transfected plasmids were integrated into the CHO DNA. Gene copy number analysis revealed that both the Ser3 1 and the Ser3.4 mutants amplifiable when grown in increasing concentrations of MTX. Retrovirus-mediated gene transfer of the Ser31 mutant into mouse marrow progenitor cells also resulted in MTX-resistant CFU-GM (colony-forming unit-granulocyte macrophage) cells.

Modulation of cytotoxicity of chemotherapeutic drugs by activated H-γas

Cells from a single MCF-7 clone were transfected with an isopropyl-1-thio-beta-D-galactopyranoside (IPTG)-inducible construct containing activated human H-ras with a Gly12 --> Val12 mutation. Expression of H-ras was induced by the presence of IPTG with low background. MCF-7-ras clones were examined for sensitivity to a wide variety of drugs under both induced and non-induced conditions. When expression of the activated ras was induced, these clones showed markedly increased resistance to cisplatin and mitomycin C, moderately increased resistance to methotrexate and trimetrexate, and no increased resistance to other drugs including taxol, doxorubicin, and etoposide. A DNA fragmentation assay revealed that DNA in MCF-7-ras cells treated with cisplatin under induced conditions was intact, whereas extensive degradation of DNA occurred in similarly treated cells under non-induced conditions. This result, along with the fact that MCF-7-ras cells, upon induction of the activated H-ras, showed increased resistance to drugs that bind DNA, indicates that the activated H-ras may play a role in the DNA repair process.

Mechanisms of Methotrexate Resistance in Acute Leukemia

Drug resistance limits the effectiveness of methotrexate (MTX) for the treatment of acute leukemia. An increased understanding of the pathways involved in folate metabolism has allowed investigations of the mechanisms of resistance observed in leukemic blasts obtained from patients. Acute lymphocytic leukemia (ALL) was studied for mechanisms of acquired MTX resistance. MTX transport in 27 patients with untreated ALL and 31 patients with relapsed ALL was measured using a previously described competitive displacement assay. Only 13% of the untreated patients were considered to have impaired MTX transport whereas over 70% of the relapsed patients had evidence of impaired MTX transport. Northern analyses and quantitative RT-PCR for the reduced folate carrier (RFC) were performed on the RNA available from the leukemic blasts of 24 patients in whom MTX transport had been measured. Six of 9 samples with impaired MTX transport had decreased RFC expression (one had no detectable RFC expression), while three had no decrease in RFC expression. Acute myelocytic leukemia (AML) was studied to determine the basis of the decreased MTX polyglutamylation. Enzyme kinetics of the enzyme folylpolyglutamate synthetase (FPGS) were studied, demonstrating FPGS in the myeloid cell lines and patient samples had a higher K(m) for MTX as a substrate than lymphoid cells. Measuring gamma-glutamyl hydrolase enzyme activity allowed a more accurate prediction of steady state levels of MTX polyglutamates. A knowledge of the mechanisms of MTX resistance that occur in leukemic blasts obtained from patients may allow the development of therapeutic strategies to circumvent resistance.

Polymerase Chain Reaction Analysis of DNA from Paraffin-Embedded Tissue

One of the greatest potentials of polymerase chain reaction (PCR) lies in the fact that even minute amounts of target DNA or extensively damaged DNA can be successfully amplified in vitro and thus become amenable to further study. This enables a detailed molecular analysis of small amounts of DNA from tissue that has been damaged by fixation (e.g., in formalin) and long-term storage in paraffin. The applications of this methodology are nearly unlimited. For example, rare tumors that are stored as formalin-fixed, paraffin-embedded tissue in pathology departments throughout the world can be analyzed at the molecular level. Furthermore, tissue from small lesions (e.g., primary skin melanomas), which are only rarely available for molecular analysis since the entire specimen is usually needed for histopathological assessment, can be examined. For PCR analysis, only several sections from the paraffin block, which are usually dispensable, are sufficient. Even small amounts of very low quality DNA can be used, as the sensitivity of the detection of specific target DNA sequences is several orders of magnitude higher than that with any conventional method. For example, Southern blot analysis of DNA from paraffin-embedded tissue has been performed, but with limited success as only relatively small amounts of degraded and irreversibly modified DNA can be obtained from embedded specimens (1,2). Thus, PCR methodology creates an ideal link between traditional histology and modern molecular biology (3).

Effects of wild‐type p53 expression on the quantity and activity of topoisomerase IIα and β 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 IIα was not altered by the transfection as determined by immunoblotting, while the amount of the β 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 IIα 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 IIα. No changes in topoisomerase IIβ immunostaining were observed. These results suggest that an epitope for topoisomerase IIα is concealed in cells expressing wild‐type p53 and that a complex between topoisomerase IIα and p53 may be disrupted by the addition of antitumor drugs. J. Cell. Biochem. 75:245–257, 1999.