The roles of homologous recombination and the immune system in the genomic evolution of cancer.

A variety of factors, whether extracellular (mutagens/carcinogens and viruses in the environment, chronic inflammation and radiation associated with the environment and/or electronic devices/machines) and/or intracellular (oxidative metabolites of food, oxidative stress due to inflammation, acid production, replication stress, DNA replication/repair errors, and certain hormones, cytokines, growth factors), pose a constant threat to the genomic integrity of a living cell. However, in the normal cellular environment multiple biological pathways including DNA repair, cell cycle, apoptosis and the immune system work in a precise, regulated (tightly controlled), timely and concerted manner to ensure genomic integrity, stability and proper functioning of a cell. If damage to DNA takes place, it is efficiently and accurately repaired by the DNA repair systems. Homologous recombination (HR) which utilizes either a homologous chromosome (in G1 phase) or a sister chromatid (in G2) as a template to repair the damage, is known to be the most precise repair system. HR in G2 which utilizes a sister chromatid as a template is also called an error free repair system. If DNA damage in a cell is so extensive that it overwhelms the repair system/s, the cell is eliminated by apoptosis. Thus, multiple pathways ensure that genome of a cell is intact and stable. However, constant exposure to DNA damage and/or dysregulation of DNA repair mechanism/s poses a risk of mutation and cancer. Oncogenesis, which seems to be a multistep process, is associated with acquisition of a number of genomic changes that enable a normal cell to progress from benign to malignant transformation. Transformed/cancer cells are recognized and killed by the immune system. However, the ongoing acquisition of new genomic changes enables cancer cells to survive/escape immune attack, evolve into a more aggressive phenotype, and eventually develop resistance to therapy. Although DNA repair (especially the HR) and the immune system play unique roles in preserving genomic integrity of a cell, they can also contribute to DNA damage, genomic instability and oncogenesis. The purpose of this article is to highlight the roles of DNA repair (especially HR) and the immune system in genomic evolution, with special focus on gastrointestinal cancer.

Nucleotide excision repair is a potential therapeutic target in multiple myeloma.

Despite the development of novel drugs, alkylating agents remain an important component of therapy in multiple myeloma (MM). DNA repair processes contribute towards sensitivity to alkylating agents and therefore we here evaluate the role of nucleotide excision repair (NER), which is involved in the removal of bulky adducts and DNA crosslinks in MM. We first evaluated NER activity using a novel functional assay and observed a heterogeneous NER efficiency in MM cell lines and patient samples. Using next-generation sequencing data, we identified that expression of the canonical NER gene, excision repair cross-complementation group 3 (ERCC3), significantly impacted the outcome in newly diagnosed MM patients treated with alkylating agents. Next, using small RNA interference, stable knockdown and overexpression, and small-molecule inhibitors targeting xeroderma pigmentosum complementation group B (XPB), the DNA helicase encoded by ERCC3, we demonstrate that NER inhibition significantly increases sensitivity and overcomes resistance to alkylating agents in MM. Moreover, inhibiting XPB leads to the dual inhibition of NER and transcription and is particularly efficient in myeloma cells. Altogether, we show that NER impacts alkylating agents sensitivity in myeloma cells and identify ERCC3 as a potential therapeutic target in MM.

Targeting homologous recombination and telomerase in Barrett's adenocarcinoma: impact on telomere maintenance, genomic instability and tumor growth.

Homologous recombination (HR), a mechanism to accurately repair DNA in normal cells, is deregulated in cancer. Elevated/deregulated HR is implicated in genomic instability and telomere maintenance, which are critical lifelines of cancer cells. We have previously shown that HR activity is elevated and significantly contributes to genomic instability in Barrett's esophageal adenocarcinoma (BAC). The purpose of this study was to evaluate therapeutic potential of HR inhibition, alone and in combination with telomerase inhibition, in BAC. We demonstrate that telomerase inhibition in BAC cells increases HR activity, RAD51 expression, and association of RAD51 to telomeres. Suppression of HR leads to shorter telomeres as well as markedly reduced genomic instability in BAC cells over time. Combination of HR suppression (whether transgenic or chemical) with telomerase inhibition, causes a significant increase in telomere attrition and apoptotic death in all BAC cell lines tested, relative to either treatment alone. A subset of treated cells also stain positive for ß-galactosidase, indicating senescence. The combined treatment is also associated with decline in S-phase and a strong G2/M arrest, indicating massive telomere attrition. In a subcutaneous tumor model, the combined treatment resulted in the smallest tumors, which were even smaller (P=0.001) than those that resulted from either treatment alone. Even the tumors removed from these mice had significantly reduced telomeres and evidence of apoptosis. We therefore conclude that although telomeres are elongated by telomerase, elevated RAD51/HR assist in their maintenance/stabilization in BAC cells. Telomerase inhibitor prevents telomere elongation but induces RAD51/HR, which contributes to telomere maintenance/stabilization and prevention of apoptosis, reducing the efficacy of treatment. Combining HR inhibition with telomerase renders telomeres more vulnerable to degradation and significantly increases/expedites their attrition, leading to apoptosis. We therefore demonstrate that a therapy targeting HR and telomerase has the potential to prevent both tumor growth and genomic evolution in BAC.

Genomic evolution in Barrett's adenocarcinoma cells: critical roles of elevated hsRAD51, homologous recombination and Alu sequences in the genome.

A prominent feature of most cancers including Barrett's adenocarcinoma (BAC) is genetic instability, which is associated with development and progression of disease. In this study, we investigated the role of recombinase (hsRAD51), a key component of homologous recombination (HR)/repair, in evolving genomic changes and growth of BAC cells. We show that the expression of RAD51 is elevated in BAC cell lines and tissue specimens, relative to normal cells. HR activity is also elevated and significantly correlates with RAD51 expression in BAC cells. The suppression of RAD51 expression, by short hairpin RNA (shRNA) specifically targeting this gene, significantly prevented BAC cells from acquiring genomic changes to either copy number or heterozygosity (P<0.02) in several independent experiments employing single-nucleotide polymorphism arrays. The reduction in copy-number changes, following shRNA treatment, was confirmed by Comparative Genome Hybridization analyses of the same DNA samples. Moreover, the chromosomal distributions of mutations correlated strongly with frequencies and locations of Alu interspersed repetitive elements on individual chromosomes. We conclude that the hsRAD51 protein level is systematically elevated in BAC, contributes significantly to genomic evolution during serial propagation of these cells and correlates with disease progression. Alu sequences may serve as substrates for elevated HR during cell proliferation in vitro, as they have been reported to do during the evolution of species, and thus may provide additional targets for prevention or treatment of this disease.

Telomerase inhibitor GRN163L inhibits myeloma cell growth in vitro and in vivo.

Human telomerase, the reverse transcriptase which extends the life span of a cell by adding telomeric repeats to chromosome ends, is expressed in most cancer cells but not in the majority of normal somatic cells. Inhibition of telomerase therefore holds great promise as anticancer therapy. We have synthesized a novel telomerase inhibitor GRN163L, a lipid-attached phosphoramidate oligonucleotide complementary to template region of the RNA subunit of telomerase. Here, we report that GRN163L is efficiently taken up by human myeloma cells without any need of transfection and is resistant to nucleolytic degradation. The exposure of myeloma cells to GRN163L led to an effective inhibition of telomerase activity, reduction of telomere length and apoptotic cell death after a lag period of 2-3 weeks. Mismatch control oligonucleotides had no effect on growth of myeloma cells. The in vivo efficacy of GRN163L was confirmed in two murine models of human multiple myeloma. In three independent experiments, significant reduction in tumor cell growth and better survival than control mice was observed. Furthermore, GRN163L-induced myeloma cell death could be significantly enhanced by Hsp90 inhibitor 17AAG. These data provide the preclinical rationale for clinical evaluation of GRN163L in myeloma and in combination with 17AAG.

Biological pathways and in vivo antitumor activity induced by Atiprimod in myeloma.

Atiprimod (Atip) is a novel oral agent with anti-inflammatory properties. Although its in vitro activity and effects on signaling in multiple myeloma (MM) have been previously reported, here we investigated its molecular and in vivo effects in MM. Gene expression analysis of MM cells identified downregulation of genes involved in adhesion, cell-signaling, cell cycle and bone morphogenetic protein (BMP) pathways and upregulation of genes implicated in apoptosis and bone development, following Atip treatment. The pathway analysis identified integrin, TGF-beta and FGF signaling as well as Wnt/beta-catenin, IGF1 and cell-cycle regulation networks as being most modulated by Atip treatment. We further evaluated its in vivo activity in three mouse models. The subcutaneous model confirmed its in vivo activity and established its dose; the SCID-hu model using INA-6 cells, confirmed its ability to overcome the protective effects of BM milieu; and the SCID-hu model using primary MM cells reconfirmed its activity in a model closest to human disease. Finally, we observed reduced number of osteoclasts and modulation of genes related to BMP pathways. Taken together, these data demonstrate the in vitro and in vivo antitumor activity of Atip, delineate potential molecular targets triggered by this agent, and provide a preclinical rational for its clinical evaluation in MM.

Expression of AAV Rep proteins in SV40-transformed and untransformed cells: reciprocal interaction with host DNA synthesis.

Adeno-associated virus (AAV) inhibits the induction of host DNA synthesis by simian virus 40 (SV40) large-tumour (T) antigen, mediated through AAV-encoded 'Rep' regulatory proteins. Rep proteins are normally synthesized by AAV-infected cells only in the presence of adenovirus. However, we observed a low level of Rep protein expression in SV40 transformed cells even in the absence of helper virus. In an effort to understand the functional interaction between SV40 T antigen and regulators of AAV rep expression, we evaluated Rep protein production by cell lines transformed with various T antigen mutants known to vary in their induction of host DNA synthesis. We observed Rep protein expression proportional to SV40-induced host DNA synthesis, as measured previously for these T antigen mutants in the absence of AAV, suggesting that rep gene expression - although it opposes the oncogenic stimulation of cell cycling by SV40 - may itself be elicited by host DNA synthesis. To test this, we employed two inhibitors of DNA synthesis: hydroxyurea, which acts by depleting deoxyribose nucleotide triphosphate pools, and aphidicolin, a specific inhibitor of DNA polymerases alpha and delta. Each inhibitor markedly and significantly reduced Rep protein levels, both in immortal cells transformed by wild-type T antigen and in normal human fibroblasts, confirming the dependence of Rep protein expression on host DNA synthesis.

Dual level inhibition of E2F-1 activity by adeno-associated virus Rep78.

E2F-1, a major cellular transcription factor, plays a pivotal role in regulating the cell cycle. The activity of E2F-1 is negatively regulated by its interaction with retinoblastoma protein (pRB), and disruption of the pRB-E2F-1 complex, a hallmark of cellular transformation by DNA tumor viruses, leads to cell proliferation. Adeno-associated virus-2 (AAV) is known to have onco-suppressive properties against DNA tumor viruses. Here we provide, for the first time, the molecular basis for antioncogenic activity of AAV. Rep78, a major regulatory protein of AAV, interacts at the protein level with E2F-1 and stabilizes the pRB-E2F-1 complex. At the DNA level, Rep78 binds to a putative site on the E2F-1 promoter and down-regulates the adenovirus-induced E2F-1 transcription. This dual level of Rep78 activity leads to decreased cellular levels of free E2F-1, leading to its onco-suppressive properties.

The placenta in meconium staining: lesions and early neonatal outcome.

PURPOSE: To evaluate the immediate neonatal outcome and the presence of various placental lesions in 96 pregnancies with meconium-stained amniotic fluid. MATERIALS AND METHODS: The patients were divided into a group with acute (N = 41) and subacute and chronic (N = 55) meconium staining of the placenta. Apgar scores, arterial cord pH and admission to the neonatal intensive care unit (NICU) were determined in addition to the findings on gross and microscopic examination of the placentas. RESULTS: Of the 53 live births with subacute and chronic meconium staining, 13% had Apgar Scores < or = 7 at 5 minutes compared to 7% with acute meconium staining. Similarly, a significantly lower umbilical artery pH was determined in the former group [(32%) versus (7%)], (p < 0.01). When 9 different pathologic lesions of the placenta were evaluated microscopically, the frequency of villous vascular thrombosis (25.4%), infarcts (38%), acute chorioamnionitis (20%), villous edema (9.1%) and villitis (14.5%) was significantly higher in the group with longer meconium exposure compared to the other group (2.4%), (9.7%), (7.3%), (0%), and 1 (2.4%), respectively. In addition, when tested for 4 different lesions, cases with acute meconium were less likely to have one or more lesions. When one or more placental lesions were found, NICU admission rate was significantly higher in the patients with subacute and chronic meconium. CONCLUSION: Subacute and chronic meconium discharge is associated with significant placental lesions and an increased risk of adverse pregnancy outcome in the immediate neonatal period.

Telomerase inhibition by peptide nucleic acids reverses 'immortality' of transformed human cells.

Telomerase activity, the ability to add telomeric repeats to the ends of chromosomes, has been detected in most immortal cell lines including tumor cells, but is low or absent in most diploid, mortal cells such as those of somatic tissues. Peptide nucleic acids (PNAs), analogs of DNA or RNA which bind to complementary nucleic acids with very high affinity, were co-electroporated into immortal human cells along with a selectable plasmid. Introduction of PNAs inverse-complementary to telomerase RNA effectively inhibited telomerase activity in intact cells, shortened telomeres, reduced colony size, and arrested cell proliferation after a lag period of 5-30 cell generations, consistent with suppression of their 'immortality'. Electroporation of selection plasmid alone had no effect, while PNAs of altered sequence were markedly less effective in each assay. This constitutes the first demonstration of cell growth arrest through telomerase inhibition, upon treatment of intact cells with an exogenous compound which can be efficiently delivered in vivo. The phenotype of telomerase-inhibited transformed cells differs from senescence of normal diploid fibroblasts, but rather resembles the crisis state of incompletely transformed cells.

Interaction of adeno-associated virus Rep78 with p53: implications in growth inhibition.

Adeno-associated virus (AAV) is a nonpathogenic, single-stranded DNA virus belonging to the parvoviridae family. Onco-suppressive properties of AAV against adenovirus, a DNA tumor virus, have been well documented. Rep78, a major regulatory protein of AAV, is believed to be responsible for its antioncogenic properties. Most DNA tumor viruses disturb the cell cycle pathways by essentially abrogating the functions of p53. Here we present evidence that AAV acts as an antiproliferative agent against adenovirus by protecting the adenoviral-mediated degradation of p53 as confirmed by both Western blot analysis and immunoprecipitation analysis with anti-p53 antibody. Coimmunoprecipitation experiments revealed that the AAV Rep78 is physically bound to p53 in vivo. Furthermore, the binding of purified p53 to the AAV Rep78 affinity column confirms their interaction. These results document for the first time that the antiproliferative effects of AAV against adenovirus are mediated, at least in part, by the interaction of AAV Rep78 with p53.

Recombination and its roles in DNA repair, cellular immortalization and cancer.

Genetic recombination is the creation of new gene combinations in a cell or gamete, which differ from those of progenitor cells or parental gametes. In eukaryotes, recombination may occur at mitosis or meiosis. Mitotic recombination plays an indispensable role in DNA repair, which presumably directed its early evolution; the multiplicity of recombination genes and pathways may be best understood in this context, although they have acquired important additional functions in generating diversity, both somatically (increasing the immune repertoire) and in germ line (facilitating evolution). Chromosomal homologous recombination and HsRad51 recombinase expression are increased in both immortal and preimmortal transformed cells, and may favor the occurrence of multiple oncogenic mutations. Tumorigenesis in vivo is frequently associated with karyotypic instability, locus-specific gene rearrangements, and loss of heterozygosity at tumor suppressor loci - all of which can be recombinationally mediated. Genetic defects which increase the rate of somatic mutation (several of which feature elevated recombination) are associated with early incidence and high risk for a variety of cancers. Moreover, carcinogenic agents appear to quite consistently stimulate homologous recombination. If cells with high recombination arise, either spontaneously or in response to "recombinogens," and predispose to the development of cancer, what selective advantage could favor these cells prior to the occurrence of growth-promoting mutations? We propose that the augmentation of telomere-telomere recombination may provide just such an advantage, to hyper-recombinant cells within a population of telomerase-negative cells nearing their replicative (Hayflick) limit, by extending telomeres in some progeny cells and thus allowing their continued proliferation.

Elevated recombination in immortal human cells is mediated by HsRAD51 recombinase.

Normal diploid cells have a limited replicative potential in culture, with progressively increasing interdivision time. Rarely, cell lines arise which can divide indefinitely; like tumor cells, such "immortal" lines display frequent chromosomal aberrations which may reflect high rates of recombination. Recombination frequencies within a plasmid substrate were 3.5-fold higher in nine immortal human cell lines than in six untransformed cell strains. Expression of HsRAD51, a human homolog of the yeast RAD51 and Escherichia coli recA recombinase genes, was 4.5-fold higher in immortal cell lines than in mortal cells. Stable transformation of human fibroblasts with simian virus 40 large T antigen prior to cell immortalization increased both chromosomal recombination and the level of HsRAD51 transcripts by two- to fivefold. T-antigen induction of recombination was efficiently blocked by introduction of HsRAD51 antisense (but not control) oligonucleotides spanning the initiation codon, implying that HsRAD51 expression mediates augmented recombination. Since p53 binds and inactivates HsRAD51, T-antigen-p53 association may block such inactivation and liberate HsRAD51. Upregulation of HsRAD51 transcripts in T-antigen-transformed and other immortal cells suggests that recombinase activation can also occur at the RNA level and may facilitate cell transformation to immortality.

Induction of duplication reversion in human fibroblasts, by wild-type and mutated SV40 T antigen, covaries with the ability to induce host DNA synthesis.

Intrachromosomal homologous recombination, manifest as reversion of a 14-kbp duplication in the hypoxanthine phosphoribosyl transferase (HPRT) gene, is elevated in human cells either stably transformed or transiently transfected by the SV40 (simian virus 40) large T antigen gene. Following introduction of wild-type SV40, or any of several T-antigen point mutations in a constant SV40 background, we observed a strong correlation between the stimulation of chromosomal recombination and induction of host-cell DNA synthesis. Moreover, inhibitors of DNA replication (aphidicolin and hydroxyurea) suppress SV40-induced homologous recombination to the extent that they suppress DNA synthesis. Stable integration of plasmids encoding T antigen also augments homologous recombination, which is suppressed by aphidicolin. We infer that the mechanism by which T antigen stimulates homologous recombination in human fibroblasts involves DNA replicative synthesis.

Expression of SV40 large T antigen stimulates reversion of a chromosomal gene duplication in human cells.

Transformation of human cells is characterized by altered cell morphology, frequent karyotypic abnormalities, reduced dependence on growth factors and substrate, and rare "immortalization"-clonal acquisition of unlimited proliferative potential. We previously reported a marked increase in DNA rearrangements, arising between two duplicated segments in a transfected plasmid substrate, for five immortal human cell lines relative to three normal fibroblast strains [Finn et al. (1989) Mol. Cell. Biol. 9, 4009-4017]. We have now assessed reversion of a 14-kilobase-pair duplication within the hypoxanthine phosphoribosyl transferase (HPRT) gene locus, in a fibroblast strain during its normal replicative lifespan and after stable transformation with SV40 large-T antigen. Revertants, selected under HPRT-dependent growth conditions immediately after purging preexisting HPRT+ cells, were confirmed as HPRT+ by hypoxanthine incorporation and 6-thioguanine sensitivity. Southern blot analyses indicate loss from most revertant clones of a restriction fragment representing the duplicated HPRT region, as predicted for homologous recombination between the 14-kilobase-pair repeats. Amplification of a subregion of HPRT mRNA implicated deletion of duplicated exons in 93% of revertant colonies. Reversion to HPRT+ was unaltered during the normal in vitro lifespan of these cells, but increased in 9 clones stably transformed with large-T antigen (mean = 3.8-fold; each P < 10(-5)). Stimulation of HPRT-reversion is abrogated in a variety of T-antigen mutants, and depends on continued induction of T antigen by glucocorticoid in two clones tested 10-30 doublings before replicative senescence. Since no immortal subclones arose from these clones, elevated reversion must precede immortalization. Increased DNA rearrangements, in cells expressing T-antigen, could facilitate the rare concurrence of multiple mutations necessary for immortalization.

Reduced telomere length in ataxia-telangiectasia fibroblasts.

Chromosomal instability with a high frequency of telomere fusion is characteristic of ataxia-telangiectasia cells both in vivo and in vitro. We have measured telomere length and found it to be consistently reduced in both diploid and SV40-transformed cells A-T fibroblasts, relative to control cells. We examined a few possible mechanisms which might account for telomeric length reduction, including telomerase activity in transformed cells and endogenous nuclease activities, but found no differences between A-T and control cells in these parameters.

Defining genes that govern longevity in Caenorhabditis elegans.

We previously identified five regions on the chromosomal map of Caenorhabditis elegans, containing genes that help specify life span in this species, by comparing the genotypes of young and long-lived progeny from a cross between strains Bristol-N2 and Bergerac-BO [Ebert et al. (1993): Genetics 135:1003-1010]. Analyses of additional crosses, and of putative polymorphisms for the implicated genes, are necessary to clarify the roles of naturally occurring polymorphic alleles in determining longevity. We therefore carried out a second multigenerational cross, between strains Bristol-N2 and DH424 (both nonmutators at 20 degrees C), to create a different heterogeneous recombinant-inbred population. We again found strong evidence implicating multiple genes, which differ between the parental strains, in the determination of life span. Increased variance of survival, for F2 and homozygous F25 worms relative to F1 hybrids, is consistent with such alleles assorting randomly in the cross progeny. Moreover, chromosome mapping data corroborate the polygenic nature of this quantitative trait. Genotypes of young and very long-lived adult worms from a synchronous F15 population were determined by polymerase chain reaction, to identify the parental strain of origin for each of 10 polymorphic loci. Two regions, on chromosomes II and IV, each contain at least one gene with allelic differences in associated longevity. A recombinant-inbred Bergerac-BO x Bristol-N2 population, derived from the earlier cross between those strains, was exposed to an acute toxic level of hydrogen peroxide. Genotyping of H2O2-resistant worms implicated at least one of the five chromosomal regions previously identified in the same cross progeny as harboring a longevity-determining gene. Superoxide dismutase and catalase levels, determined for the three parental strains as they aged, confirm the existence of polymorphisms in the corresponding genes (or their regulatory mechanisms) inferred from the chromosome-II mapping data, and are consistent with the hypothesis that increased longevity is conferred by high levels of these enzymes late in life.