Immune checkpoint inhibitors for POLE or POLD1 proofreading-deficient metastatic colorectal cancer.

BACKGROUND: POLE and POLD1 proofreading deficiency (POLE/D1pd) define a rare subtype of ultramutated metastatic colorectal cancer (mCRC; over 100 mut/Mb). Disease-specific data about the activity and efficacy of immune checkpoint inhibitors (ICIs) in POLE/D1pd mCRC are lacking and it is unknown whether outcomes may be different from mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) mCRCs treated with ICIs. PATIENTS AND METHODS: In this global study, we collected 27 patients with mCRC harboring POLE/D1 mutations leading to proofreading deficiency and treated with anti-programmed cell death-ligand 1 alone +/- anti-cytotoxic T-lymphocyte antigen-4 agents. We collected clinicopathological and genomic characteristics, response, and survival outcomes after ICIs of POLE/D1pd mCRC and compared them with a cohort of 610 dMMR/MSI-H mCRC patients treated with ICIs. Further genomic analyses were carried out in an independent cohort of 7241 CRCs to define POLE and POLD1pd molecular profiles and mutational signatures. RESULTS: POLE/D1pd was associated with younger age, male sex, fewer RAS/BRAF driver mutations, and predominance of right-sided colon cancers. Patients with POLE/D1pd mCRC showed a significantly higher overall response rate (ORR) compared to dMMR/MSI-H mCRC (89% versus 54%; P = 0.01). After a median follow-up of 24.9 months (interquartile range: 11.3-43.0 months), patients with POLE/D1pd showed a significantly superior progression-free survival (PFS) compared to dMMR/MSI-H mCRC [hazard ratio (HR) = 0.24, 95% confidence interval (CI) 0.08-0.74, P = 0.01] and superior overall survival (OS) (HR = 0.38, 95% CI 0.12-1.18, P = 0.09). In multivariable analyses including the type of DNA repair defect, POLE/D1pd was associated with significantly improved PFS (HR = 0.17, 95% CI 0.04-0.69, P = 0.013) and OS (HR = 0.24, 95% CI 0.06-0.98, P = 0.047). Molecular profiling showed that POLE/D1pd tumors have higher tumor mutational burden (TMB). Responses were observed in both subtypes and were associated with the intensity of POLE/D1pd signature. CONCLUSIONS: Patients with POLE/D1pd mCRC showed more favorable outcomes compared to dMMR/MSI-H mCRC to treatment with ICIs in terms of tumor response and survival.

CHK1 Inhibition Is Synthetically Lethal with Loss of B-Family DNA Polymerase Function in Human Lung and Colorectal Cancer Cells.

Checkpoint kinase 1 (CHK1) is a key mediator of the DNA damage response that regulates cell-cycle progression, DNA damage repair, and DNA replication. Small-molecule CHK1 inhibitors sensitize cancer cells to genotoxic agents and have shown single-agent preclinical activity in cancers with high levels of replication stress. However, the underlying genetic determinants of CHK1 inhibitor sensitivity remain unclear. We used the developmental clinical drug SRA737 in an unbiased large-scale siRNA screen to identify novel mediators of CHK1 inhibitor sensitivity and uncover potential combination therapies and biomarkers for patient selection. We identified subunits of the B-family of DNA polymerases (POLA1, POLE, and POLE2) whose silencing sensitized the human A549 non-small cell lung cancer (NSCLC) and SW620 colorectal cancer cell lines to SRA737. B-family polymerases were validated using multiple siRNAs in a panel of NSCLC and colorectal cancer cell lines. Replication stress, DNA damage, and apoptosis were increased in human cancer cells following depletion of the B-family DNA polymerases combined with SRA737 treatment. Moreover, pharmacologic blockade of B-family DNA polymerases using aphidicolin or CD437 combined with CHK1 inhibitors led to synergistic inhibition of cancer cell proliferation. Furthermore, low levels of POLA1, POLE, and POLE2 protein expression in NSCLC and colorectal cancer cells correlated with single-agent CHK1 inhibitor sensitivity and may constitute biomarkers of this phenotype. These findings provide a potential basis for combining CHK1 and B-family polymerase inhibitors in cancer therapy. SIGNIFICANCE: These findings demonstrate how the therapeutic benefit of CHK1 inhibitors may potentially be enhanced and could have implications for patient selection and future development of new combination therapies.

DNA Polymerases as targets for gene therapy of hepatocellular carcinoma.

BACKGROUND: Hepatocyte carcinoma (HCC) is one of the most common malignancies worldwide. Despite many achievements in diagnosis and treatment, HCC mortality remains high due to the malignant nature of the disease. Novel approaches, especially for targeted therapy, are being extensively explored. Gene therapy is ideal for such purpose for its specific expression of exogenous genes in HCC cells driven by tissue-specific promoter. However strategies based on correction of mutations or altered expressions of genes responsible for the development/progression of HCC have limitations because these aberrant molecules are not presented in all cancerous cells. In the current work, we adopted a novel strategy by targeting the DNA replication step which is essential for proliferation of every cancer cell. METHODS: A recombinant adenovirus with alpha fetoprotein (AFP) promoter-controlled expressions of artificial microRNAs targeting DNA polymerases α, δ, ε and recombinant active Caspase 3, namely Ad/AFP-Casp-AFP-amiR, was constructed. RESULTS: The artificial microRNAs could efficiently inhibit the expression of the target polymerases in AFP-positive HCC cells at both RNA and protein levels, and HCC cells treated with the recombinant virus Ad/AFP-Casp-AFP-amiR exhibited significant G0/1 phase arrest. The proliferation of HCC cells were significantly inhibited by Ad/AFP-Casp-AFP-amiR with increased apoptosis. On the contrary, the recombinant adenovirus Ad/AFP-Casp-AFP-amiR did not inhibit the expression of DNA polymerases α, δ or ε in AFP-negative human normal liver cell HL7702, and showed no effect on the cell cycle progression, proliferation or apoptosis. CONCLUSIONS: Inhibition of DNA polymerases α, δ and ε by AFP promoter-driven artificial microRNAs may lead to effective growth arrest of AFP-positive HCC cells, which may represent a novel strategy for gene therapy by targeting the genes that are essential for the growth/proliferation of cancer cells, avoiding the limitations set by any of the individually altered gene.

Lower nucleotide excision repair capacity in newborns compared to their mothers: a pilot study.

Recognition of the potential vulnerability of children and newborns and protection of their health is essential, especially regarding to genotoxic compounds. Benzo(a)pyrene B(a)P a commonly found carcinogen, and its metabolite BPDE, are known to cross the placenta. To investigate how well newborns are able to cope with BPDE-induced DNA damage, a recent developed nucleotide excision repair cell phenotype assay was applied in a pilot study of 25 newborn daughters and their mothers, using the Alkaline Comet Assay and taking demographic data into account. Newborns seemed to be less able to repair BPDE-induced DNA damage since lower repair capacity levels were calculated compared to their mothers although statistical significance was not reached. Assessment of repair capacity in combination with genotypes will provide important information to support preventive strategies in neonatal care and to define science based exposure limits for pregnant women and children.

Downregulation of peroxiredoxin V stimulates formation of etoposide-induced double-strand DNA breaks.

Antioxidant protein Peroxiredoxin V (PrxV) is located in mitochondria and peroxisomes but is also present in the nucleus. Here, we show that nuclear PrxV associates with coilin-containing bodies suggesting possible interaction of this protein with transcription complexes. We also studied etoposide-induced phosphorylation of histone H2AX (gamma-H2AX) in human cells in which PrxV activity was downregulated (knockdown, KD-clones) or compromised by overexpression of redox-negative (RD) protein. In KD clones, but not in RD-clones, formation of etoposide-induced gamma-H2AX was increased, indicating that PrxV inhibits conversion of topoisomerase II cleavage complexes into double-strand DNA breaks but this inhibition is not caused by its antioxidant activity.

L-Homoserylaminoethanol, a novel dipeptide alcohol inhibitor of eukaryotic DNA polymerase from a plant cultured cells, Nicotina tabacum L.

We found a novel inhibitor specific to eukaryotic DNA polymerase epsilon(pol epsilon) from plant cultured cells, Nicotina tabacum L. The compound (compound 1) was a dipeptide alcohol, L-homoserylaminoethanol. The 50% inhibition of pol epsilon activity by the compound was 43.6 microg/mL, and it had almost no effect on the activities of the other eukaryotic DNA polymerases such as alpha, beta, gamma and delta, prokaryotic DNA polymerases, nor DNA metabolic enzymes such as human telomerase, human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase, human DNA topoisomerase I and II, T4 polynucleotide kinase and bovine deoxyribonuclease I. Kinetic studies showed that inhibition of pol epsilon by the compound was non-competitive with respect to both template-primer DNA and nucleotide substrate. We succeeded in chemically synthesizing the stereoisomers, L-homoserylaminoethanol and D-homoserylaminoethanol, and found both were effective to the same extent. The IC(50) values of L- and D-homoserylaminoethanols for pol epsilon were 42.0 and 41.5 microg/mL, respectively. This represents the second discovery of a pol epsilon-specific inhibitor, and the first report on a water-soluble peptide-like compound as the inhibitor, which is required in biochemical studies of pol epsilon.

Noncompetitive counteractions of DNA polymerase epsilon and ISW2/yCHRAC for epigenetic inheritance of telomere position effect in Saccharomyces cerevisiae.

Relocation of euchromatic genes near the heterochromatin region often results in mosaic gene silencing. In Saccharomyces cerevisiae, cells with the genes inserted at telomeric heterochromatin-like regions show a phenotypic variegation known as the telomere-position effect, and the epigenetic states are stably passed on to following generations. Here we show that the epigenetic states of the telomere gene are not stably inherited in cells either bearing a mutation in a catalytic subunit (Pol2) of replicative DNA polymerase epsilon (Pol epsilon) or lacking one of the nonessential and histone fold motif-containing subunits of Pol epsilon, Dpb3 and Dpb4. We also report a novel and putative chromatin-remodeling complex, ISW2/yCHRAC, that contains Isw2, Itc1, Dpb3-like subunit (Dls1), and Dpb4. Using the single-cell method developed in this study, we demonstrate that without Pol epsilon and ISW2/yCHRAC, the epigenetic states of the telomere are frequently switched. Furthermore, we reveal that Pol epsilon and ISW2/yCHRAC function independently: Pol epsilon operates for the stable inheritance of a silent state, while ISW2/yCHRAC works for that of an expressed state. We therefore propose that inheritance of specific epigenetic states of a telomere requires at least two counteracting regulators.

Pyridoxal 5'-phosphate is a selective inhibitor in vivo of DNA polymerase alpha and epsilon.

Vitamin B(6) compounds such as pyridoxal 5(')-phosphate (PLP), pyridoxal (PL), pyridoxine (PN), and pyridoxamine (PM), which reportedly have anti-angiogenic and anti-cancer effects, were thought to be inhibitors of some types of eukaryotic DNA polymerases. PL moderately inhibited only the activities of calf DNA polymerase alpha (pol alpha), while PN and PM had no inhibitory effects on any of the polymerases tested. On the other hand, PLP, a phosphated form of PL, was potentially a strong inhibitor of pol alpha and epsilon from phylogenetic-wide organisms including mammals, fish, insects, plants, and protists. PLP did not suppress the activities of prokaryotic DNA polymerases such as Escherichia coli DNA polymerase I and Taq DNA polymerase, or DNA-metabolic enzymes such as deoxyribonuclease I. For pol alpha and epsilon, PLP acted non-competitively with the DNA template-primer and competitively with the nucleotide substrate. Since PL was converted to PLP in vivo after being incorporated into human cancer cells, the anti-angiogenic and anti-cancer effects caused by PL must have been caused by the inhibition of pol alpha and epsilon activities after conversion to PLP.

A sulphoquinovosyl diacylglycerol is a DNA polymerase epsilon inhibitor.

Sulphoquinovosyl diacylglycerol (SQDG) was reported as a selective inhibitor of eukaryotic DNA polymerases alpha and beta [Hanashima, Mizushina, Ohta, Yamazaki, Sugawara and Sakaguchi (2000) Jpn. J. Cancer Res. 91, 1073-1083] and an immunosuppressive agent [Matsumoto, Sahara, Fujita, Shimozawa, Takenouchi, Torigoe, Hanashima, Yamazaki, Takahashi, Sugawara et al. (2002) Transplantation 74, 261-267]. The purpose of this paper is to elucidate the biochemical properties of the inhibition more precisely. As expected, SQDG could inhibit the activities of mammalian DNA polymerases such as alpha, delta, eta and kappa in vitro in the range of 2-5 micro M, and beta and lambda in vitro in the range of 20-45 micro M. However, SQDG could inhibit only mammalian DNA polymerases epsilon (pol epsilon) activity at less than 0.04 micro M. SQDG bound more tightly to mammalian pol epsilon than the other mammalian polymerases tested. Moreover, SQDG could inhibit the activities of all the polymerases from animals such as fish and insect, but not of the polymerases from plant and prokaryotes. SQDG should, therefore, be called a mammalian pol epsilon-specific inhibitor or animal polymerase-specific inhibitor. To our knowledge, this represents the first report about an inhibitor specific to mammalian pol epsilon.

Enhanced nucleotide excision repair in cisplatin resistant human KB carcinoma cells.

We previously reported that enhanced active efflux of cisplatin and increased GSH level were observed in KCP-4 cells. In the present study, KCP-4 cells were found to be cross-resistant to ultraviolet (UV) compared with parental KB-3-1 cells. Enhanced nucleotide excision repair (NER) was verified by time-dependent repair of UV-induced DNA damage. In addition, the amount of platinum bound to DNA after exposure to cisplatin decreased in a time-dependent manner in KCP-4 cells and this was reversed by aphidicolin, a DNA polymerase inhibitor. In stationary phase cultures, aphidicolin increased the sensitivity of KCP-4 cells to cisplatin. The expression of xeroderma pigmentosum complementation group F (XPF), an endonuclease involved in NER, was upregulated in KCP-4 cells. In KCP-4 cells the expression of hMSH6, one of the mismatch repair (MMR) factors, was decreased compared to parental KB-3-1 and revertant KCP-4R cells. However, KCP-4 cells were cross-resistant to oxaliplatin, and microsatellite instability was not observed in them. These findings suggest that the enhanced NER activity for DNA damage caused by cisplatin may be involved in cisplatin resistance in KCP-4 cells.

Novel phenalenone derivatives from a marine-derived fungus exhibit distinct inhibition spectra against eukaryotic DNA polymerases.

A number of compounds used for cancer chemotherapy exert their effects by inhibiting DNA replication. New inhibitors of DNA polymerases, therefore, could be potential candidates for new anti-cancer drugs. This study tested the effects of two phenalenone-skeleton-based compounds, which were isolated from a marine-derived fungus Penicillium sp., sculezonone-B (SCUL-B) and sculezonone-A (SCUL-A), upon DNA polymerase activity. Both compounds inhibited bovine DNA polymerases alpha and gamma, moderately affected the activity of DNA polymerase epsilon, and had almost no effect on HIV-reverse transcriptase and an E. coli DNA polymerase I Klenow fragment. Most notably, whereas SCUL-A inhibited pol beta (IC(50) = 17 microM), SCUL-B has only a weak influence upon this polymerase (IC(50) = 90 microM). Kinetic studies showed that inhibition of both DNA polymerases alpha and beta by either SCUL-A or SCUL-B was competitive with respect to dTTP substrate and noncompetitive with the template-primer. Whereas pol alpha inhibition by SCUL-B is competitive with respect to dATP, the inhibition by SCUL-A was found to be a mixed type with dATP substrate. The K(i) values of SCUL-B were calculated to be 1.8 and 6.8 microM for DNA polymerases alpha and gamma, respectively. The K(i) of DNA polymerase beta for SCUL-A was 12 microM and that for DNA polymerase alpha, 16 microM. Therefore, deletion of the OH-group at C12 enhanced inhibition of DNA polymerase beta. Since computational analyses of these two inhibitors revealed a remarkable difference in the distribution of negative electrostatic charge on the surface of molecules, we infer that different electrostatic charges might elicit different inhibition spectra from these two compounds.

Mode of inhibition of HIV-1 reverse transcriptase by polyacetylenetriol, a novel inhibitor of RNA- and DNA-directed DNA polymerases.

Polyacetylenetriol (PAT), a natural marine product from the Mediterranean sea sponge Petrosia sp., was found to be a novel general potent inhibitor of DNA polymerases. It inhibits equally well the RNA- and DNA-dependent DNA polymerase activities of retroviral reverse transcriptases (RTs) (i.e. of HIV, murine leukaemia virus and mouse mammary tumour virus) as well as cellular DNA polymerases (i.e. DNA polymerases alpha and beta and Escherichia coli polymerase I). A study of the mode and mechanism of the polymerase inhibition by PAT has been conducted with HIV-1 RT. PAT was shown to be a reversible non-competitive inhibitor. PAT binds RT independently and at a site different from that of the primer-template and dNTP substrates with high affinity (K(i)=0.51 microM and K(i)=0.53 microM with dTTP and with dGTP as the variable substrates respectively). Blocking the polar hydroxy groups of PAT has only a marginal effect on the inhibitory capacity, thus hydrophobic interactions are likely to play a major role in inhibiting RT. Preincubation of RT with the primer-template substrate prior to the interaction with PAT reduces substantially the inhibition capacity, probably by preventing these contacts. PAT does not interfere with the first step of polymerization, the binding of RT to DNA, nor does the inhibitor interfere with the binding of dNTP to RT/DNA complex, as evident from the steady-state kinetic study, whereby K(m) remains unchanged. We assume, therefore, that PAT interferes with subsequent catalytic steps of DNA polymerization. The inhibitor may alter the optimal stereochemistry of the polymerase active site relative to the primer terminus, bound dNTP and the metal ions that are crucial for efficient catalysis or, alternatively, may interfere with the thumb sub-domain movement and, thus, with the translocation of the primer-template following nucleotide incorporation.

Anatomy of the primase-alpha DNA polymerase reaction accomplished by nucleoprotein complexes harboring an extrachromosomal DNA identical with avian myeloblastosis virus core-bound DNA: influencing by carbonyldiphosphonate, mimosine and butylphenyl deoxyguanosine-5'-triphosphate.

Activities of the primase (Pr)-alpha DNA polymerase (pol) enzyme complex belonging to the naturally occurring reaction systems represented by special NP complexes harboring an extrachromosomal DNA identical with avian myeloblastosis virus (AMV) core-bound DNA (J. Riman, A. Sulová and K. Horská, Acta virol. 39, 149-159 (1995); J. Ríman and A. Sulová, Acta virol. 41, 181-192 (1997)) were studied in the absence and presence of carbonyldiphosphonate (COMDP), mimosine (MIMO), to it related ciclopirox olamine (CPX) and butylphenyl deoxyguanosine-5'-triphosphate (BuPdGTP). Reaction products radioactively labeled for RNA and DNA and synthesized with the common four ribonucleoside triphosphates (rNTPs) or rNTPs and deoxyribonucleoside triphosphates (dNTPs) in the reaction medium, were analyzed by polyacrylamide gel electrophoresis (PAGE) at denaturing conditions. It was shown that COMDP strongly activates the Pr and uncouples its activity from that of alpha DNA pol with accumulation of initiator (i) RNAs of the basic length. This phenomenon is not affected by BuPdGTP. MIMO, in contrast, stimulates both pol activities of this enzyme complex and preserves their mutual coupling. The effects of COMDP, MIMO and CPX seem to be modulated by concentration of the ambient dNTPs. Addition of dNTPs to rNTPs makes the effects of COMDP and MIMO mutually exclusive, suggesting that both these agents, though chemically quite different, are competing for one active site responsible for coupling these both pol activities into the one Pr-alpha DNA pol reaction.

A neutralizing antibody against human DNA polymerase epsilon inhibits cellular but not SV40 DNA replication.

The contribution of human DNA polymerase epsilon to nuclear DNA replication was studied. Antibody K18 that specifically inhibits DNA polymerase activity of human DNA polymerase epsilon in vitro significantly inhibits DNA synthesis both when microinjected into nuclei of exponentially growing human fibroblasts and in isolated HeLa cell nuclei. The capability of this neutralizing antibody to inhibit DNA synthesis in cells is comparable to that of monoclonal antibody SJK-132-20 against DNA polymerase alpha. Contrary to the antibody against DNA polymerase alpha, antibody K18 against DNA polymerase epsilon did not inhibit SV40 DNA replication in vitro. These results indicate that DNA polymerase epsilon plays a role in replicative DNA synthesis in proliferating human cells like DNA polymerase alpha, and that this role for DNA polymerase epsilon cannot be modeled by SV40 DNA replication.

Activities of a lagging DNA strand synthesis of nucleoprotein complexes harboring an extrachromosomal DNA closely related to avian myeloblastosis virus core-bound DNA.

Nucleoprotein (NP) complexes constituting the material of the postmicrosomal sediment (POMS) and its three basic components (A, B, C) (Ríman and Sulová, 1997a), harboring an extrachromosomal DNA closely related to AMV DNA were found to possess DNA- and RNA-synthesizing activities (SAs) reflecting the ability of this material to be intensely labelled for DNA and RNA, respectively. The types of these NA-SAs were compatible with those significant for a lagging DNA strand synthesis (LSS). The use of selective inhibitors and of the proliferating cell nuclear antigen (PCNA) disclosed a successive involvement of alpha DNA polymerase (pol) and PCNA-insensitive delta DNA pol in LSS. In this respect, we show gradual changes in the representation of activities (As) of both mentioned DNA pols in the NP complexes of the individual POMS components. Those of POMS component C contained alpha DNA pol As only, while a distinct portion of DNA SAs of POMS component B was represented on expense of alpha DNA pol As by PCNA-insensitive delta DNA pol (epsilon DNA pol), As which represented practically all the DNA SAs of POMS component A. The type of RNA SAs of this material represented mostly by primase (Pr) As corresponded well with the nature of LSS. An exception was represented by a minor portion of RNA-SAs of POMS component A which was alpha amanitin-sensitive like RNA pol II. Moreover, analyzing this natural model replication system, we found that the carbonyldiphosphonate (COMDP), a selective inhibitor of the PCNA-insensitive delta DNA pol, was a strong activator of Pr-As and/or Pr-alpha DNA pol As of NP complexes of POMS component C.

Products of a lagging DNA strand synthesis of nucleoprotein complexes harboring an extrachromosomal DNA closely related to avian myeloblastosis virus core-bound DNA.

Nucleoprotein (NP) complexes present in selected fractions of the separated three basic components A, B, C of the postmicrosomal sediment (POMS) (Ríman and Sulová, 1997a) were used as a source of nucleic acid synthesizing activities (NA-SAs) expressed in reactions in vitro. These were performed in the absence and presence of N2-(p-butylphenyl) deoxyguanosine 5'-triphosphate (BuPdGTP), aphidicolin (Aph) and carbonyldiphosphonate (COMDP), inhibitors allowing differentiation between two DNA polymerases (pols) involved in a lagging DNA strand synthesis (LSS). Reaction products were isolated and analyzed by polyacrylamide gel electrophoresis (PAGE) in denaturing conditions. Products labelled with [alpha-32P]dAMP or [alpha-32P]AMP were represented by intermediates significant for LSS. Okazaki fragment precursors, whose synthesis was inhibited by BuPdGTP and resistant to Aph, and whose radioactive RNA label was DNase I-sensitive, represented products formed in vitro by NP complexes of POMS component C. Okazaki fragments 127 b in length, whose synthesis was insensitive to BuPdGTP but inhibited by Aph and COMDP, were characteristic of the reactions accomplished by NP complexes of POMS component B while products of NA-SAs of NP complexes of POMS component A were represented by Okazaki fragments up to 280 b in length, whose synthesis was most sensitive to Aph. In accord with previous data (Ríman and Sulová, 1997b), COMDP strongly stimulated production of RNAs corresponding in length with initiator RNAs (iRNAs). However, in dependence on reaction conditions also ribodeoxyribonucleotide primers can be produced by NP complexes of POMS component C, suggesting the occurrence of two primase (Pr) catalytic modes influenced by dNTP/rNTP relation and thus, by COMDP, a strong competitor for dNTPs. These results represent the first direct evidence that an extrachromosomal DNA organized into special NP complexes can be replicated extrachromosomally by a mechanism of LSS.

KGF facilitates repair of radiation-induced DNA damage in alveolar epithelial cells.

Administration of exogenous keratinocyte growth factor (KGF) prevents or attenuates several forms of oxidant-mediated lung injury. Because DNA damage in epithelial cells is a component of radiation pneumotoxicity, we determined whether KGF ameliorated DNA strand breaks in irradiated A549 cells. Cells were exposed to 137Cs gamma rays, and DNA damage was measured by alkaline unwinding and ethidium bromide fluorescence after a 30-min recovery period. Radiation induced a dose-dependent increase in DNA strand breaks. The percentage of double-stranded DNA after exposure to 30 Gy increased from 44.6 +/- 3.5% in untreated control cells to 61.6 +/- 5.0% in cells cultured with 100 ng/ml KGF for 24 h (P < 0.05). No reduction in DNA damage occurred when the cells were cultured with KGF but maintained at 0 degree C during and after irradiation. The sparing effect of KGF on radiation-induced DNA damage was blocked by aphidicolin, an inhibitor of DNA polymerases-alpha, -delta, and -epsilon and by butylphenyl dGTP, which blocks DNA polymerase-alpha strongly and polymerases-delta and -epsilon less effectively. However, dideoxythymidine triphosphate, a specific inhibitor of DNA polymerase-beta, did not abrogate the KGF effect. Thus KGF increases DNA repair capacity in irradiated pulmonary epithelial cells, an effect mediated at least in part by DNA polymerases-alpha, -delta, and -epsilon. Enhancement of DNA repair capability after cell damage may be one mechanism by which KGF is able to ameliorate oxidant-mediated alveolar epithelial injury.

Rat hepatoma Reuber H-35 cells produce factors that promote the hatching of mouse embryos cultured in vitro.

The effects of coculture and conditioned medium of rat hepatoma Reuber H-35 cells on the subsequent in vitro development and hatching of mouse 2-cell embryos were examined. The hatching of embryos obtained from CD-1 mice was accelerated by coculture with Reuber H-35 cells in the presence of 3 mg/ml BSA. The promoting effect on complete hatching from zona pellucida was evident even in cell-conditioned medium containing 60 micrograms/ml BSA. In the presence of 60 micrograms/ml BSA, more than 20% of embryos completely hatched, whereas none hatched in the control culture. The promoting activity was also found in both the M(r) < 10,000 and the M(r) > 10,000 subfractions of the conditioned medium separated by ultrafiltration. The cell number per blastocyst was increased to 1.1- to 1.3 times the control by culturing embryos from the 2-cell stage with the conditioned medium or its subfractions. The effective target of promoting factors for complete hatching was after the morula stage, and blastocysts hatched completely even when incubated in conditioned medium for 6 h. Inhibitors of DNA polymerase alpha, protein synthesis, and protein kinase partially reduced (40-90% inhibition) the promoting effect of the conditioned medium. On the other hand, protease inhibitors showed no effect. In a caseinolytic assay, protease activity was undetectable in the conditioned medium. Incubating the 125I-labeled proteins derived from the M(r) > 10,000 fraction with blastocysts revealed that at least 9 proteins with apparent molecular masses of 76, 60, 49, 38, 34, 31, 24, 22, and 18 kDa specifically bound to or accumulated in the embryos. Moreover, reverse-transcriptase polymerase chain reaction showed that Reuber H-35 cells expressed mRNAs for epidermal growth factor, transforming growth factors alpha and beta 1, and stem cell factor. These results indicated that embryonic development and the process of zona hatching was accelerated by factors synthesized by Reuber H-35 cells. This and other studies demonstrated that Reuber H-35 cells exert positive (later than 2-cell stage) and negative (at 2-cell stage) effects upon the development of mouse embryos at different embryonic stages. These factors will serve as valuable tools to clarify the proliferating and differentiating mechanisms of the preimplantation embryo.

Increased efficacy of aphidicolin killing of human neuroblastoma cells in vitro by encapsulation in liposomes.

Aphidicolin is a tetracyclic diterpene antibiotic which kills human neuroblastoma cells (NB) in vitro while it has no significant effect on the viability of different human cell types including normal embryonal cells. In the present study, we tested whether aphidicolin encapsulated in liposomes kills NB cells with the efficacy superior to that of unencapsulated aphidicolin. The drug was entrapped in vesicles composed of phosphatidylcholine, phosphatidylserine and cholesterol in a molar ratio of 83:5:12. The treatment with encapsulated aphidicolin at a concentration of 200 nmol for 5 days killed all cells of three human NB cell lines. In contrast, at least 30% of the cells survived 5 days of treatment with 200 nmol unencapsulated aphidicolin. The results showed that aphidicolin killing of human NB cells may be increased by encapsulation in liposomes.

Inibiçäo seletiva das atividades das enzimas transcriptase reversa do vírus HIV-1 e DNA polimerases humanas por derivados dipirazolo-piridina / Selective inhibition of reverse transcriptase enzyme activity of HIV-1 virus and human polymerases DNA by dipirazolo-pyridines derivatives

Novos ribonucleosídeos derivados dos sistemas dipirazolo-piridina foram preparados e avaliados quanto à atividade polimerásica das enzimas transcriptase reversa (RT) do vírus HIV-1 e das DNA polimerases humanas alfa e epsilon. Os derivados 1b e 1d inibiram a atividade da transcriptase reversa em concentraçöes de micromolares. Entretanto, as mesmas substâncias näo foram capazes de inibir a atividade polimerase das enzimas DNA-polimerase humana alfa e epsilon.