William A. Franklin

Heat-Shock Proteins Associated with Base Excision Repair Enzymes in HeLa Cells

Abstract Mendez, F., Sandigursky, M., Franklin, W.A., Kenny, M.K., Kureekattil, R. and Bases, R. Heat-Shock Proteins Associated with Base Excision Repair Enzymes in HeLa Cells. Two enzymes of base excision repair (BER), uracil DNA glycosylase (UDG) and DNA polymerase β (β pol), from HeLa cells co-eluted from Superose 12 FPLC columns. The UDG was completely displaced from 150–180-kDa fractions to 30–70-kDa fractions by brief treatment with 0.5 N NaCl, pH 3.0, as expected when protein–protein associations are disrupted, but β pol was not displaced by this treatment. UDG was not essential to the presence of β pol in the 150–180-kDa enzyme complex. β pol and UDG apparently reside in separate but co-eluting structures. Immunoaffinity chromatography showed that the association of UDG and β pol was accounted for by attachment in common to DNA and that the association was abolished by eliminating DNA. Evidence for base excision repairosomes containing UDG and β pol in protein–protein assemblies was not found. However, UDG and human AP endonuclease (HAP1) were associated with HSP70 and HSP27, which are present in 150–180-kDa and 30–70-kDa proteins of cell sonicates. The association of HSPs with BER enzymes was confirmed by hydroxyl radical protein–protein footprinting and immunoaffinity tests. The association of HSPs and BER enzymes is a novel finding. HSP binding may account for the presence of BER enzymes in the two large size class fractions and HSPs may have functional roles in BER.

Radiation- and Heat-Induced Apoptosis in PC-3 Prostate Cancer Cells

Radiation therapy is used to treat all stages of localized prostate cancer. However, both clinical and radiobiological evidence indicate that prostate cancer cells are relatively resistant to radiation. New modalities which can induce apoptosis in androgen-independent cells are of interest for developing novel treatments for prostate cancer. Both radiation and hyperthermia have been shown to be able to induce apoptosis in a variety of cells. However, it is not known whether heat- or radiation-induced apoptosis plays a role in the mechanism of cell killing for prostate cancer cells. We examined radiation- and heat-induced apoptosis in PC-3 prostate cancer cells and found that apoptosis is an important mode of death in heated cells. However, apoptosis is not an important mechanism of death in irradiated cells. Apoptosis-inducing modalities such as hyperthermia may supplement radiation therapy in the future management of prostate cancer. No significant apoptosis was observed when cells were heated at 42 degrees C for 240 min. Thus a heating temperature of 43 degrees C and above may be required to induce significant apoptosis in a clinically feasible duration of time.

Protein-Protein Interactions between the Escherichia coli Single-Stranded DNA-Binding Protein and Exonuclease I

It was demonstrated previously that a deoxyribophosphodiesterase (dRpase) activity is associated with the DNA repair enzyme exonuclease I, and that this activity is stimulated by the addition of the E. coli single-stranded DNA-binding protein (Ssb). This activity catalyzes the release of deoxyribose-phosphate groups at apurinic/apyrimidinic (AP) sites in the DNA that have been cleared by the action of an AP endonuclease. We have now used the yeast two-hybrid system to demonstrate that a protein-protein interaction occurs between exonuclease I and Ssb. When the E. coli ssb gene was fused in frame to the DNA-activating domain of the GAL4 transcriptional activator and the exonuclease I gene was fused in frame to the DNA-binding domain, a functional GAL4 transcriptional activator was produced as determined by growth of yeast on selective medium and the measurement of beta-galactosidase activity. We have also demonstrated that Ssb can stimulate the dRpase activity of exonuclease I using double-stranded bacteriophage M13 DNA containing several strand interruptions at incised AP sites. These results suggest that Ssb may be required for efficient base-excision repair in bacteria.

DNA Deoxyribophosphodiesterase and an Activity That Cleaves DNA Containing Thymine Glycol Adducts in Deinococcus radiodurans

Deinococcus radiodurans is the most radioresistant bacterium discovered to date. Recently it has been demonstrated that this organism contains the DNA repair enzyme uracil-DNA glycosylase and an apurinic/apyrimidinic (AP) endonuclease that may function as part of a DNA base excision repair pathway. We demonstrate here that a DNA deoxyribophosphodiesterase activity that acts on incised AP sites in DNA to remove deoxyribose-phosphate groups is found in lysates prepared from D. radiodurans cells. The partially purified activity was found to be smaller in size than the E. coli dRpase activity, with an estimated molecular weight of 25-30 kDa. In addition, an activity that recognizes and cleaves DNA containing thymine glycols was also detected, with a molecular weight of approximately 30 kDa. This enzyme may be analogous to the thymine glycol glycosylase/AP lyase endonuclease III of E. coli.

7-Hydroxystaurosporine (UCN-01) Preferentially Sensitizes Cells with a Disrupted TP53 to Gamma Radiation in Lung Cancer Cell Lines

Abstract Xiao, H. H., Makeyev, Y., Butler, J., Vikram, B. and Franklin, W. A. 7-Hydroxystaurosporine (UCN-01) Preferentially Sensitizes Cells with a Disrupted TP53 to Gamma Radiation in Lung Cancer Cell Lines. Radiat. Res. 158, 84–93 (2002). Mutations in TP53 occur in more than 50% of the lung cancer patients and are associated with an increased resistance to chemotherapy and radiotherapy. The human lung adenocarcinoma cell lines A549 and LXSN contain a wild-type TP53 and were growth arrested at both the G1- and G2-phase checkpoints after irradiation. However, a TP53-disrupted cell line, E6, was arrested only at the G2-phase checkpoint. UCN-01 (7-hydroxystaurosporine), a CHEK1 inhibitor that abrogates the G2 block, has been reported to enhance radiation toxicity in human lymphoma and colon cancer cell lines. In this study, UCN-01 preferentially enhanced the radiosensitivity of the TP53-disrupted E6 cells compared to the TP53 wild-type cells. This effect was more pronounced in cells synchronized in early G1 phase, where the E6 cells showed a higher resistance to radiation in the absence of drug. These results indicate that the combination of UCN-01 and radiation can more specifically target resistant TP53 mutated cancer cells and spare TP53 wild-type normal cells.

Excision of sugar-phosphate products at apurinic/apyrimidinic sites by DNA deoxyribophosphodiesterase of Escherichia coli.

It has been shown previously that the DNA deoxyribophosphodiesterase (dRpase) activity of Escherichia coli excises 2-deoxyribose 5-phosphate moieties at apurinic/apyrimidinic (AP) sites in DNA following cleavage of the DNA at the AP site by an AP endonuclease such as endonuclease IV of E coli. A second class of enzymes that cleave DNA at AP sites by a beta-elimination mechanism, AP lyases, leave a different sugar-phosphate product remaining at the AP site, which has been identified as the compound trans-4-hydroxy-2-pentenal 5-phosphate. It is shown that dRpase removes this unsaturated sugar-phosphate group following cleavage of a poly(dA-dT) substrate containing AP sites by the action of the AP lyase endonuclease III of E. coli. The Km for the removal of trans-4-hydroxy-2-pentenal 5-phosphate is 0.06 microM; the Km for the removal of 2-deoxyribose 5-phosphate is 0.17 microM. It was verified that the sugar-phosphate product removed by dRpase from the endonuclease III-cleaved substrate was trans-4-hydroxy-2-pentenal 5-phosphate by conversion of the product to the compound cyclopentane-1,2-dione. The dRpase activity is unique in its ability to remove sugar-phosphate products after cleavage by both AP endonucleases and AP lyases.