Heat shock protein 70 binds to human apurinic/apyrimidinic endonuclease and stimulates endonuclease activity at abasic sites.

The interaction of human heat shock protein 70 (HSP70) with human apurinic/apyrimidinic endonuclease (HAP1) was demonstrated by coimmunoprecipitation. A combination of HSP70 and HAP1 also caused a shift in the electrophoretic mobility of a DNA fragment containing an apurinic/apyrimidinic site. The functional consequence of the HSP70/HAP1 interaction was a 10-100-fold enhancement of endonuclease activity at abasic sites. The physical and functional interaction between HSP70 and HAP1 did not require the addition of ATP. The association of HSP70 and a key base excision repair enzyme suggests a role for heat shock proteins in promoting base excision repair. These findings provide a possible mechanism by which HSP70 protects cells against oxidative stress.

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 beta (beta 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 beta pol was not displaced by this treatment. UDG was not essential to the presence of beta pol in the 150-180-kDa enzyme complex. beta pol and UDG apparently reside in separate but co-eluting structures. Immunoaffinity chromatography showed that the association of UDG and beta 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 beta 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.

Accelerated regression of brain metastases in patients receiving whole brain radiation and the topoisomerase II inhibitor, lucanthone.

PURPOSE: To determine if lucanthone crossed the blood-brain barrier in experimental animals; and to determine accelerated tumor regression of human brain metastases treated jointly with lucanthone and whole brain radiation. METHODS AND MATERIALS: The organ distribution of 3H lucanthone in mice and 125I lucanthone in rats was determined to learn if lucanthone crossed the blood-brain barrier. Size determinations were made of patients' brain metastases from magnetic resonance images or by computed tomography before and after treatment with 30 Gy whole brain radiation alone or with lucanthone. RESULTS: The time course of lucanthone's distribution in brain was identical to that in muscle and heart after intraperitoneal or intravenous administration in experimental animals. Lucanthone, therefore, readily crossed the blood-brain barrier in experimental animals. CONCLUSION: Compared with radiation alone, the tumor regression in patients with brain metastases treated with lucanthone and radiation was accelerated, approaching significance using a permutation test at p = 0.0536.

Topoisomerase inhibition by lucanthone, an adjuvant in radiation therapy.

PURPOSE: To determine whether lucanthone can inhibit human topoisomerases in vitro. METHODS AND MATERIALS: Lucanthone was incubated with human topoisomerases II and I together with their plasmid substrates, to determine if lucanthone interfered with the catalytic activities of topoisomerases and if it enhanced the formation of DNA strand breaks, as determined by agarose gel electrophoresis of the resultant plasmid forms. RESULTS: Incubation of the enzymes with lucanthone inhibited the catalytic activity of topoisomerases II and I. With topoisomerase II, it increased the abundance of DNA double strand breaks (cleavable complexes). CONCLUSION: Lucanthone, like actinomycin D, inhibited topoisomerases II and I. It may act to enhance the yield of DNA double strand breaks in cells through a mechanism of topoisomerase II inhibition.

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.

Evaluation of 2-(3-aminobenzamido)-2-deoxy-D-glucose as a radiosensitizer.

PURPOSE: 3-Aminobenzamide (3-AB), an inhibitor of poly(adenosine diphosphate [ADP]-ribose) synthetase, functions as a radiosensitizer in several human tumor cell lines. 2-(3-AB)-2-deoxy-D-glucose (3-AB-G) was designed to increase preferentially the intracellular concentration of the drug in tumor cells. Both the toxicity and effectiveness of 3-AB-G as a radiosensitizer were determined. MATERIALS AND METHODS: The toxicity of 3-AB-G was measured in HeLa and Chinese hamster ovary cells. The radiosensitizing effect of 3-AB-G was determined for both cell lines. RESULTS: 3-AB-G was not toxic to cells at concentrations of 10 mmol/L or less. 3-AB-G did not alter cell survival after irradiation. CONCLUSION: 3-AB-G was not an effective radiosensitizer for the cells tested. Coupling 2-deoxyglucose to 3-AB may block the uptake of the inhibitor into the cell by altering the ability of the receptor to recognize the molecule or may interfere with the specificity of the inhibitor for poly(ADP-ribose) synthetase.

Enhanced repair endonuclease activities from radiation-arrested G2 phase mammalian cells.

HeLa cells arrested in G2 phase 22 h after receiving 11.5 Gy gamma-radiation contained 3.6-fold more EDTA-resistant DNA repair endonuclease activity than unirradiated cells. Enzyme activity was determined by measuring the release of fragments from an irradiated repetitive alpha DNA substrate or from synthetic substrates containing a single modified base, 8-oxoguanine (8-oxo-G), a major radiation product. It appeared that the radiation-induced enhanced repair activity in some cells might be a feature of radiation-induced G2 arrest. Indeed, unirradiated G2 HeLa cells that had been synchronized by double thymidine block contained 3-7-fold more endonuclease activity than G1 or S-phase cells. Similarly, two of four other cell lines tested exhibited elevated repair endonuclease activity in G2. However, all six cell lines tested exhibited radiation-enhanced repair endonuclease activity. Therefore, the underlying mechanism for radiation enhancement of enzyme activity remains to be clarified and does not seem to be completely accounted for as a consequence of G2 arrest. The results showed different substrate specificities among cell lines as well as differences during the cell cycle of individual cell lines. Repair endonuclease activity from all cell lines which we have tested were associated with 60-70 kDa proteins from Superose 12 columns. Since reports from other laboratories have described several different DNA repair activities in 50-70 kDa Superose 12 fractions, it seems possible that the DNA repair enzymes may be associated in a repairosome structure.

Enhanced excision repair activity in mammalian cells after ionizing radiation.

Monkey CV-1 cells which had received 5 Gy 12 h before harvesting lysates from their cell cultures contained approximately three times as much DNA excision repair enzyme activity as unirradiated cells. The activity was determined in crude cell lysates by the release of intermediate mobility DNA fragments and fragments with 3'-phosphoryl ends from 5'-32P-end labelled irradiated 95 bp alpha DNA. Different 3'-termini endow the fragments with differing mobilities, signifying steps in the processing of radiation damaged DNA. Similar results were obtained when Krebs II mouse tumour cells growing in mice as ascites received 5 Gy 12 h before harvest. The enzyme activities from CV-1 cells and from Krebs II cells were partially purified as 60-70 kDa proteins on Superose 12 or Ultrogel AcA-54 columns. Divalent cations were not required for enzyme activity. A 23 nucleotide long defined duplex oligodeoxynucleotide substrate containing a single 8-oxodG residue was also very actively cleaved by the partially purified cell enzymes. 8-oxoguanine is a major product of ionizing radiation's action on DNA and was recognized by the enzymes described here. The mechanism by which radiation increased excision repair activity of cellular enzymes is not understood.

Repair of ionizing radiation damage in primate alpha DNA transfected into rat cells.

The time-course of repair of irradiated primate alpha DNA was studied after transfection and recovery from rat NRK cells. Rat cells were chosen for transfection because they have no alpha DNA. Plasmid pBUC4 alpha 10, containing 10 tandem 172 bp alpha DNA subunits in its 5 kbp DNA, was irradiated and introduced into the rat cells by electroporation. The transfected alpha DNA was then recovered from NRK nuclei free of extraneous rat DNA, permitting study of the fate of the transfected alpha DNA in time-course experiments. alpha DNA continuously entered nuclei for processing in the first 2.5 h after transfection. The pool of damaged bases in alpha DNA in NRK nuclei was detectable 2.5 h after transfection. Radiation-induced alpha DNA fragments of electrophoretic mobility intermediate between those of unit nucleotide length were prominent in sequencing gel analyses of alpha DNA for 5-150 min after transfection. These intermediate mobility fragments initially disappeared with T 1/2 of 6-20 min. The alpha DNAs of intermediate mobility are presumed to be intermediates in DNA repair. Residual DNA base damage which had not been processed in the transfected cells could later be unmasked in vitro by conversion to strand breaks by beta-elimination using heat and piperidine or endonuclease III of E. coli. Irradiation of the recipient NRK cells with 5 Gy 4 hours before transfection prolonged the time during which intermediate mobility species could be found, consistent with the increased frequency of intermediate mobility species observed in DNA of monkey CV-1 cells pretreated with small doses of radiation before 300 Gy (Bases et al. 1990).

DNA base and strand damage in X-irradiated monkey CV-1 cells: influence of pretreatment using small doses of radiation.

Base damage in alpha DNA from irradiated monkey CV-1 cells was determined by measuring release of 5'-32P-end labelled DNA fragments after digestion with endonuclease III of E. coli. The frequency and base sequence locations of the enzyme-sensitive sites were determined. Fragments were released from irradiated DNA at sequence sites of pyrimidines and guanines. The time for repair of half the single strand breaks was approximately 1.5 h. Repair of base damage as judged from loss of enzyme-sensitive sites in DNA was slower, with more than half of the damaged bases still detectable after 4 h of repair. Two important changes in the pattern of fragment release from DNA were produced when small radiation doses preceded the large ones needed to produce measurable DNA strand breaks and base damage. 5 Gy to cells incubated several hours before 320 Gy increased by five-fold the abundance of small DNA fragments with 3'-phosphoryl termini detected in high-resolution denaturing gels. These increases were detectable with doses as small as 0.2 Gy and were accompanied by the appearance of new species of DNA fragments of intermediate mobility at specific locations in the base sequence. The patterns resemble those produced by digesting DNA from heavily irradiated cells with endonuclease III.

Base sequence damage in DNA from X-irradiated monkey CV-1 cells.

Two kinds of 3'-ends were detected in DNA scission fragments of highly repetitive primate component alpha DNA which were isolated from irradiated monkey CV-1 cells. The fragments' 3'-ends were characterized by 5'-32P-end labelling the DNA, followed by examination in high-resolution polyacrylamide gels under denaturing conditions. Hydrolysis of the labelled fragments' termini with exonuclease III of E. coli or by the 3'-phosphatase activity of T4 polynucleotide kinase generated a third, slowest migrating species in each mobility size class. Reference to mobility size class standards makes it highly probable that the fragment ends generated by X-rays in cells are 3'-phosphoryl and 3'-phosphoglycolate, and that they are converted to slower migrating fragments with 3'-OH ends, similar to results obtained with DNA irradiated in water (Henner et al. 1982, 1983 a, b). Densitometer measurements of gel autoradiograms showed that X-ray induction of DNA fragments with 3'-phosphoryl and 3'-phosphoglycolate ends was dose-dependent over a range 100-900 Gy. In CV-1 cells the frequency of single-strand breaks in alpha DNA was 8.6 x 10(-7) breaks/nt/Gy. The two kinds of ends disappeared in post-radiation incubation with a half-time of 1.6 h. These results provide a new means to study X-ray damage and repair of specific sequences in animal cells.

Expression of prokaryotic genes after transfection of X-irradiated plasmids into primate cells.

Expression of the prokaryotic gene for chloramphenicol acetyltransferase (EC 2.3.1.28) (CAT) in primate cells transfected with X-irradiated plasmid pSV2CAT was determined in transient expression assays. CAT expression did not depend upon the presence of supercoiled plasmids, but relaxed circular forms were essential. X-ray conversion of relaxed circles to linear forms paralleled the loss of CAT expression, with identical D0's in the first part of dose-response curves. X-ray-induced loss of supercoiled forms was complete at much lower doses. The D0 for inactivation of CAT expression by X irradiation of the plasmids in 1 mM Tris buffer was 270 Gy; it was 13 Gy for plasmids irradiated in water. The D0's for conversion of pSV2CAT to relaxed circle forms were only one-seventh as large as the D0's for CAT inactivation after X-ray in water or in 1 mM Tris buffer. Expression of the CAT gene in some representative repair-deficient human fibroblasts transfected with X-irradiated pSV2CAT was less than in monkey CV-1 cells or cell lines from normal human subjects. These results demonstrate a novel means to study low levels of X-ray damage in DNA correlating specific X-ray damage in the DNA with expression of the gene in unirradiated primate cells.

X-ray-induced base sequence damage in primate alphoid DNA.

Radiation-induced single-strand breaks were found throughout the 172 bp repeat units of African green monkey component alpha DNA. Two kinds of 3'-ends of 5'-32P-labeled restriction fragments were found, as previously described by others. After irradiation in vitro, the yield of single-strand breaks was 4 X 10(-5) breaks/nucleotide/Gy, as determined by analyses in DNA sequencing type gels. Protection from X-ray damage was found when the DNA received 150 Gy in the presence of 2-mercaptoethanol. The results demonstrate a very sensitive quantitative means to study the role of indirect effects of ionizing radiation on strand-break induction and protection at the base sequence level. Component alpha DNA was isolated from irradiated CV-1 cells and was analyzed for single-strand breaks. Under these conditions the frequency of breaks was less than the frequency obtained when purified DNA was irradiated. The methodology is presented because of its relevance to the study of DNA strand breakage in living cells.

IgG binding enhances DNAase I sensitivity of N-acetoxy-N-2-acetylaminofluorene-modified phi X-174 RF DNA.

DNA restriction fragments of phi X-174 RF were modified with the carcinogen, N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF). Immune complexes of 5'-32P-labeled AAF-modified DNA and rabbit immunoglobulin (IgG) against AAF-guanosine were specifically bound by surface membranes of Cowan I strain micrococci whose protein A binds the Fc portion of IgG. DNAase I sensitivity of the bound DNA was 20-fold greater than in solution, but the normal pattern of hydrolysis was not altered, as determined in sequencing gels. Nonadducted DNA ligated to AAF-modified DNA acquired the enhanced sensitivity to DNAase I hydrolysis when the ligation hybrid was immunobound.

Site specific cleavage of phi X-174 replicative form DNA after modification by N-acetoxy-N-2-acetylaminofluorene.

Three kinds of structural disturbances were found in an 88 base pair (bp) fragment of phi X-174 DNA after exposure to N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF). (i) Frequent strand scissions at two specific guanine sites on the 5' 32P-end-labeled fragment were identified by base sequence analysis. Scissions at these two sites were induced at neutral pH and they were not increased by treatment with apurinic endonuclease. They are an immediate consequence of N-Aco-AAF action and are not primarily apurinic sites. (ii) Alkali treatment with 1 M piperidine at 90 degrees C induced strand scissions at every guanine, demonstrating adduct slices, depurination and strand scissions. (iii) Adducted DNA was sensitive to single-strand specific nuclease digestion, suggesting unwound DNA. These studies indicate the prediliction of N-Aco-AAF for certain DNA sites and they suggest three kinds of DNA modifications which can be expected after adduction by this carcinogen. Some of the sites may be premutational carcinogen-induced DNA structural modifications.

An application of immunocytology to the analysis of the cell kinetics of upper respiratory and digestive tract squamous carcinoma.

An antinucleoside immunofluorescence technique (ANIF) facilitated evaluation of labeling index (LI) from frozen sections of 36 upper respiratory and digestive tract squamous cancers (URDTS) from a group of 35 patients. There were 35 URDTS of the larynx, oral cavity, or pharynx and the LIs of this population ranged from 0-39.4%, (mean, 14.7%); 6% of URDTS (2 of 36) were not assessable by ANIF. The administration of nontherapeutic radioactive materials, the establishment of cell cultures, and perturbations in cell growth implicit in the removal of tumor from host prior to assessment are unnecessary in the application of this technique.