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Perspective on This Article from Metformin Prevents Liver Tumorigenesis by Inhibiting Pathways Driving Hepatic Lipogenesis

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In the base excision repair pathway, MYH/MUTYH DNA glycosylase prevents mutations by removing adenine mispaired with 8-oxoG, a frequent oxidative lesion. MYH glycosylase activity is enhanced by Rad9-Rad1-Hus1 (9-1-1) checkpoint clamp and SIRT6 histone/protein deacetylase. Here, we show that MYH, SIRT6, and 9-1-1 are recruited to confined oxidatively damaged regions on telomeres in mammalian cells. Using different knockout cells, we show that SIRT6 responds to damaged telomeres very early, and then recruits MYH and Hus1 following oxidative stress. However, the recruitment of Hus1 to damaged telomeres is partially dependent on SIRT6. The catalytic activities of SIRT6 are not important for SIRT6 response but are essential for MYH recruitment to damaged telomeres. Compared to wild-type MYH, the recruitment of hMYHV315A mutant (defective in both SIRT6 and Hus1 interactions), but not hMYHQ324H mutant (defective in Hus1 interaction only), to damaged telomeres is severely reduced. The formation of MYH/SIRT6/9-1-1 complex is of biological significance as interrupting their interactions can increase cell's sensitivity to H2O2 and/or elevate cellular 8-oxoG levels after H2O2 treatment. Our results establish that SIRT6 acts as an early sensor of BER enzymes and both SIRT6 and 9-1-1 serve critical roles in DNA repair to maintain telomere integrity.

Prostate cancer (PCa) and breast cancer (BCa) are leading causes of cancer related deaths in American men and women, and morbidity is due to metastasis preferentially to bone. Compared to Caucasian American (CA), African American (AA) men and women have a higher bone density, as well as mortality rate due to bone metastasis. Recent studies have shown that high mobility group two (HMGA2), a non-histone chromatin binding protein, plays a critical role in promoting epithelial-mesenchymal transition (EMT) and metastasis. HMGA2 full-length/wild-type and truncated (without the 3’UTR) isoforms have been shown to be overexpressed in several cancers, however their distinct roles in metastasis have not been reported. Our laboratory focuses on tumor-micronenvironmental interactions, particularly, how PCa cells interact with bone at the metastatic site. We have previously published that PCa cells may degrade hydroxyapatite, the major inorganic component of bone, to release calcium which promotes paracrine cell proliferation and migration, and this signaling increases with increased bone density. We hypothesize that EMT is associated with prostate health disparities and that HMGA2 isoforms may play differential roles at the bone metastatic site. We examined EMT marker expression in breast and prostate patient tissue and cell lines by immunohistochemistry (IHC), RNA In Situ Hybridization (RISH), western blot, and real-time PCR. Next, LNCaP PCa cell line stably overexpressing either full length or truncated HMGA2 was co-cultured with low (100 mg) or high (200 mg) hydroxyapatite, demineralized bone matrix or human ground bone matrix, for 6 days followed by collection of condition media (CM). CM was added to parental LNCaP cells for various time points followed by analysis of paracrine cell proliferation using MTS assay, cell migration across collagen using boyden chamber, and the effect on various pathways using western blot analysis. EMT marker (Snail and Cathepsin L) protein expression was higher in AA normal and cancer tissue compared to CA, while it was intermediate in Bahamian tissue. Wild-type HMGA2 mRNA expression was higher in metastatic patient tissue compared to truncated HMGA2, and higher in AA compared to CA tissue. CM taken from LNCaP cells overexpressing wild-type and truncated HMGA2 co-cultured with hydroxyapatite, demineralized bone matrix or ground bone matrix when added to parental LNCaP cells led to higher paracrine cell proliferation and paracrine cell migration. This was associated with MAPK activation that could be abrogated by MAPK inhibitor, UO126. These results indicate that EMT is higher in AA compared to CA. Additionally, both wild-type and truncated HMGA2 interaction with bone matrix promotes paracrine cell proliferation and migration via ERK signaling. Our study shows that wild-type and truncated isoforms of HMGA2 mediate PCa/bone interactions which may lead to increased bone metastasis, particularly in African American men that have higher bone density. Citation Format: Taaliah Campbell, Ohuod Hawsawi, Bor-Jang Hwang, Liza J. Burton, Quentin Loyd, Veronica Henderson, Simone Howard, Camille Ragine, Robin Roberts, Andrew Gachii, Valerie Odero-Marah. Basic science mechanisms associated with bone tumor microenvironment and health disparities in breast and prostate cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr IA041.

People of African descent suffer more from cancer compared to other racial groups in terms of morbidity and mortality. Surveillance, Epidemiology, and End Results (SEER) reports Black men and women to have the highest diagnoses and highest cancer death rate. The reported cancer disparity statistic is also reflected by cancer type; prostate cancer was shown to be one of the top causes of cancer death in all cancer types, and the second leading cause of death, after lung cancer, in men. Men of African Descent, however, have more than a 2-fold increased mortality rate of prostate cancer compared to other ancestral populations. Cancer is considered a genetic disease, and prostate cancer has a heritability of 58%. Most studies however, do not capture the genetic heterogeneity found in men of African Descent, nor histological and morphological profiles that would provide prognostic and diagnostic markers for targeted and personalized therapeutic treatment options. Visium Spatial Transcriptomics is a method that maps transcriptomics data to the location within the tissue region that is being analyzed. Instead of being limited to a small piece of DNA or RNA, spatial transcriptomics enables high-resolution assessment of spatial gene expression across tissue sections. This technology assiduously probes the transcriptome of tissues samples in an untargeted way. Spatial Transcriptomics involves cutting edge technology that provides great potential to capture the cellular landscape of men with the heaviest burden of prostate cancer. The Visium platform by 10X Genomics is able to interrogate more than 10,000 transcripts per capture region. The protocol involves a comprehensive process from tissue collection to next generation sequencing following a bioinformatics pipeline. The complete protocol from beginning to end requires a collaboration of clinicians, pathologists, basic scientists, and bioinformaticians. This means that access to resources is imperative to the success of this technique. Unfortunately, low resource institutions are at a disadvantage in being able to perform the entire protocol. This fact is yet another type of health disparity. This observation, however, provides great opportunities to establish collaborations with other institutions. Closing the gap between higher and lower resource investigations would be vital in addressing prostate cancer health disparity using spatial transcriptomics. I will share my observation, learning, experimentation, and joint participation effort experience with Visium Spatial Transcriptomics in my quest to understand the genetic architecture of men of African descent that has them at a prolific risk for prostate cancer. Citation Format: Maxine S. Harlemon, Ana C. Millena, Bor-Jang Hwang, Valerie Odero-Marah. Utilizing Visium spatial transcriptomics to investigate prostate cancer disparity in men of African descent [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr IA025.

High mobility group A2 (HMGA2), a non-histone protein, is known to promote epithelial-mesenchymal transition (EMT), which plays a critical role in prostate cancer progression and metastasis. Both full-length HMGA2 (WT-HMGA2) and truncated (lacking the 3’UTR) HMGA2 (TR-HMGA2) isoforms are overexpressed in several cancers such as lung carcinomas, ovarian cancer, breast cancer, and gastric cancer. However, there is no study investigating the expression and differential roles of WT vs truncated HMGA2 isoforms in prostate cancer. Our previous results indicated that the overexpression of HMGA2 in LNCaP cells increased cell viability and migration for both wild-type and truncated HMGA2. Promotion of EMT was observed in wild-type, but not truncated HMGA2. Reactive oxygen species (ROS) levels were increased with truncated HMGA2 more than wild-type HMGA2. Therefore, wild-type and truncated HMGA2 may play different roles on cancer progression and metastasis. The goal of this study is to examine the expression of wild-type vs. truncated HMGA2 in prostate cancer patient tissue. We have developed a way to detect location and expression of wild-type and truncated HMGA2 using RNA in situ hybridization (RISH). The specific probes’ hybridization followed by a serial signal amplification process allows us to locate and quantify the HMGA2 isoforms in Formalin-Fixed Paraffin-Embedded (FFPE) tissue specimens from cancer patients. Several prostate tissue samples and a prostate cancer tissue microarray that includes 35 patients from different races, stages of cancer, and metastasis status was analyzed. Our results showed increased HMGA2 expression (both wild-type and truncated) in several prostate cancer samples with higher stage cancer. We also observed that most wild-type and truncated HMGA2 is located within the nucleus, while some wild-type isoforms were detected within the cytoplasm. We will further quantify the expression levels to examine whether they are associated with tumor stage and/or race. These studies are the first to examine HMGA2 isoform expression and localization in prostate patient tissue and may offer novel therapeutic intervention strategies based on HMGA2 isoform expression. Citation Format: Bor-Jang Hwang, Mojisoluwa Awolowo, Taaliah Campbell, Ohuod Hawsawi, Sharon Harrison, Denise Gibbs, Camille Ragin, Valerie Odero-Marah. Detection of wild-type and truncated HMGA2 in prostate cancer tissues using RNA in situ hybridization [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C017.

Melanoma patients respond poorly to chemotherapies because they acquire drug resistance. Therapies that can overcome the resistance to inhibitors of the mutated BRAF protein kinase in melanoma are urgently needed. Chk1 protein kinase is a central component of the DNA damage response and plays a crucial role in controlling cell cycle progression. Analyses indicate that low mRNA expression of Chk1 is significantly associated with good overall survival of melanoma patients. To evaluate the effectiveness of Chk1 inhibitors in melanoma therapy, we have generated BRAF inhibitor (PLX4032 or vemurafenib) resistant melanoma cell lines (A375-PLX-R and WM9-PLX-R) from A375 and WM9, respectively. We observe that AKT (protein kinase B) is constitutively activated in A375-PLX-R, but not in WM9-PLX-R cells, suggesting that these cells develop resistance to PLX4032 through different mechanisms. We show that a potent and specific inhibitor of Chk1 (PF477736) is effective in reducing cell viability and colony formation of PLX4032-resistant cells. Even more impressively, PF477736 triggers PLX4032-resistant melanoma cells to regain sensitivity to the PLX4032. Mouse xenograft studies show that treating A375-PLX-R derived tumors with combined PLX4032 and PF477736 significantly reduce tumor growth. Combined treatments with PLX4032 and PF477736 reduce the levels of total Chk1 protein and alter Chk1 phosphorylation at several sites in both PLX4032 sensitive and resistant melanoma cells. Combinatorial treatments with PLX4032 and PF477736 to melanoma cells substantially induce DNA damage and cell death. Our results suggest that Chk1 inhibitors may provide new therapy options for melanoma patients.

Telomeres consist of special features and proteins to protect the ends of each chromosome from deterioration and fusion. The telomeric DNA repeats are highly susceptible to oxidative damage that can accelerate telomere shortening and affect telomere integrity. Several DNA repair factors including MYH/MUTYH DNA glycosylase, its interacting partners Rad9/Rad1/Hus1 checkpoint clamp, and SIRT6 aging regulator, are associated with the telomeres. MYH prevents C:G to A:T mutation by removing adenine mispaired with a frequent oxidative DNA lesion, 8-oxoguanine. Here, we show that hMYH knockout (KO) human HEK-293T cells are more sensitive to H2O2 treatment, have higher levels of DNA strand breaks and shorter telomeres than the control hMYH+/+ cells. SIRT6 foci increase at both the global genome and at telomeric regions in H2O2-treated hMYH+/+ cells. However, in untreated hMYH KO HEK-293T cells, SIRT6 foci only increase at the global genome, but not at the telomeric regions. In addition, the hMYH KO HEK-293T cells have increased extra-chromosomal and intra-chromosomal telomeres compared to the control cells, even in the absence of H2O2 treatment. After H2O2 treatment, the frequency of extra-chromosomal telomeres increased in control HEK-293T cells. Remarkably, in H2O2-treated hMYH KO cells, the frequencies of extra-chromosomal telomeres, intra-chromosomal telomeres, and telomere fusions are further increased. We further found that the sensitivity to H2O2 and shortened telomeres of hMYH KO cells, are restored by expressing wild-type hMYH, and partially rescued by expressing hMYHQ324H mutant (defective in Hus1 interaction only), but not by expressing hMYHV315A mutant (defective in both SIRT6 and Hus1 interactions). Thus, MYH interactions with SIRT6 and Hus1 are critical for maintaining cell viability and telomeric stability. Therefore, the failure to coordinate 8-oxoG repair is detrimental to telomere integrity.

Cell cycle checkpoints provide surveillance mechanisms to activate the DNA damage response, thus preserving genomic integrity. The heterotrimeric Rad9–Rad1–Hus1 (9–1–1) clamp is a DNA damage response sensor and can be loaded onto DNA. 9–1–1 is involved in base excision repair (BER) by interacting with nearly every enzyme in BER. Here, we show that individual 9–1–1 components play distinct roles in BER directed by MYH DNA glycosylase. Analyses of Hus1 deletion mutants revealed that the interdomain connecting loop (residues 134–155) is a key determinant of MYH binding. Both the N-(residues 1–146) and C-terminal (residues 147–280) halves of Hus1, which share structural similarity, can interact with and stimulate MYH. The Hus1K136A mutant retains physical interaction with MYH but cannot stimulate MYH glycosylase activity. The N-terminal domain, but not the C-terminal half of Hus1 can also bind DNA with moderate affinity. Intact Rad9 expressed in bacteria binds to and stimulates MYH weakly. However, Rad91−266 (C-terminal truncated Rad9) can stimulate MYH activity and bind DNA with high affinity, close to that displayed by heterotrimeric 91−266–1–1 complexes. Conversely, Rad1 has minimal roles in stimulating MYH activity or binding to DNA. Finally, we show that preferential recruitment of 91−266–1–1 to 5′-recessed DNA substrates is an intrinsic property of this complex and is dependent on complex formation. Together, our findings provide a mechanistic rationale for unique contributions by individual 9–1–1 subunits to MYH-directed BER based on subunit asymmetry in protein–protein interactions and DNA binding events.

The RAD9A-HUS1-RAD1 (9-1-1) complex is a heterotrimeric clamp that promotes checkpoint signaling and repair at DNA damage sites. In this study, we elucidated HUS1 functional residues that drive clamp assembly, DNA interactions, and downstream effector functions. First, we mapped a HUS1-RAD9A interface residue that was critical for 9-1-1 assembly and DNA loading. Next, we identified multiple positively charged residues in the inner ring of HUS1 that were crucial for genotoxin-induced 9-1-1 chromatin localization and ATR signaling. Finally, we found two hydrophobic pockets on the HUS1 outer surface that were important for cell survival after DNA damage. Interestingly, these pockets were not required for 9-1-1 chromatin localization or ATR-mediated CHK1 activation but were necessary for interactions between HUS1 and its binding partner MYH, suggesting that they serve as interaction domains for the recruitment and coordination of downstream effectors at damage sites. Together, these results indicate that, once properly loaded onto damaged DNA, the 9-1-1 complex executes multiple, separable functions that promote genome maintenance.

Hepatic gluconeogenesis is crucial to maintain normal blood glucose during periods of nutrient deprivation. Gluconeogenesis is controlled at multiple levels by a variety of signal transduction and transcriptional pathways. However, dysregulation of these pathways leads to hyperglycemia and type 2 diabetes. While the effects of various signaling pathways on gluconeogenesis are well established, the downstream signaling events repressing gluconeogenic gene expression are not as well understood. The cell-cycle regulator cyclin D1 is expressed in the liver, despite the liver being a quiescent tissue. The most well-studied function of cyclin D1 is activation of cyclin-dependent kinase 4 (CDK4), promoting progression of the cell cycle. We show here a novel role for cyclin D1 as a regulator of gluconeogenic and oxidative phosphorylation (OxPhos) gene expression. In mice, fasting decreases liver cyclin D1 expression, while refeeding induces cyclin D1 expression. Inhibition of CDK4 enhances the gluconeogenic gene expression, whereas cyclin D1–mediated activation of CDK4 represses the gluconeogenic gene-expression program in vitro and in vivo. Importantly, we show that cyclin D1 represses gluconeogenesis and OxPhos in part via inhibition of peroxisome proliferator–activated receptor γ coactivator-1α (PGC1α) activity in a CDK4-dependent manner. Indeed, we demonstrate that PGC1α is novel cyclin D1/CDK4 substrate. These studies reveal a novel role for cyclin D1 on metabolism via PGC1α and reveal a potential link between cell-cycle regulation and metabolic control of glucose homeostasis.

MutY DNA glycosylase homologs (MYH or MUTYH) reduce G:C to T:A mutations by removing misincorporated adenines or 2-hydroxyadenines paired with guanine or 7,8-dihydro-8-oxo-guanine (8-oxoG). Mutations in human the MYH (hMYH) gene are associated with the colorectal cancer predisposition syndrome MYH-associated polyposis. To examine the function of MYH in human cells, we regulated MYH gene expression by knockdown or overproduction. The hMYH knockdown human HeLa cells are more sensitive to the killing effects of H2O2 than the control cells. In addition, hMYH knockdown cells have altered cell morphology, display enhanced susceptibility to apoptosis, and have altered DNA signaling activation in response to oxidative stress. The cell cycle progression of hMYH knockdown cells is also different from that of the control cells following oxidative stress. Moreover, hMYH knockdown cells contain higher levels of 8-oxoG lesions than the control cells following H2O2 treatment. Although MYH does not directly remove 8-oxo-G, MYH may generate favorable substrates for other repair enzymes. Overexpression of mouse MYH (mMYH) in human mismatch repair defective HCT15 cells makes the cells more resistant to killing and refractory to apoptosis by oxidative stress than the cells transfected with vector. In conclusion, MYH is a vital DNA repair enzyme that protects cell from oxidative DNA damage and is critical for proper cellular response to DNA damage.

MutY homologue (MYH) is a DNA glycosylase which excises adenine paired with the oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG, or G(o)) during base excision repair (BER). Base excision by MYH results in an apurinic/apyrimidinic (AP) site in the DNA where the DNA sugar-phosphate backbone remains intact. A key feature of MYH activity is its physical interaction and coordination with AP endonuclease I (APE1), which subsequently nicks DNA 5' to the AP site. Because AP sites are mutagenic and cytotoxic, they must be processed by APE1 immediately after the action of MYH glycosylase. Our recent reports show that the interdomain connector (IDC) of human MYH (hMYH) maintains interactions with hAPE1 and the human checkpoint clamp Rad9-Rad1-Hus1 (9-1-1) complex. In this study, we used NMR chemical shift perturbation experiments to determine hMYH-binding site on hAPE1. Chemical shift perturbations indicate that the hMYH IDC peptide binds to the DNA-binding site of hAPE1 and an additional site which is distal to the APE1 DNA-binding interface. In these two binding sites, N212 and Q137 of hAPE1 are key mediators of the MYH/APE1 interaction. Intriguingly, despite the fact that hHus1 and hAPE1 both interact with the MYH IDC, hHus1 does not compete with hAPE1 for binding to hMYH. Rather, hHus1 stabilizes the hMYH/hAPE1 complex both in vitro and in cells. This is consistent with a common theme in BER, namely that the assembly of protein-DNA complexes enhances repair by efficiently coordinating multiple enzymatic steps while simultaneously minimizing the release of harmful repair intermediates.

Arsenic is an environmental pollutant with carcinogenic properties that is found in many regions of the world but which poses a health risk primarily in economically disadvantaged areas. In these areas, arsenic ingestion affects various tissues but particularly skin in which it acts as a comutagen with the ultraviolet component of solar radiation. Both epidemiological and experimental evidence indicates that both arsenic and ultraviolet radiation act on signaling pathways that effect the expression of cyclin D1. We have previously employed an in vitro model system of human epidermal keratinocytes to study the effects of submicormolar concentrations of sodium arsenite on cyclin D1 expression. Here, we employed this system to demonstrate concordant cyclin D1-related induction profiles of ultraviolet B radiation and arsenite using cDNA microarray analysis. We also show that both of these agents act epigenetically to bring about demethylation of the cyclin D1 promoter.

We show that a single low-dose exposure of human epidermal keratinocytes (NHEK) to an FS20 light source in vitro can induce the formation of mitochondrial DNA deletions in a PCR detection assay. We used primer sets specifically designed to exclude amplification of segments containing the common deletion, but which could detect possibly lower abundance deletions generated within the same region of the mitochondrial genome. We characterized eight novel deletions of which six were generated from cut sites within, or adjacent to, short direct repeats. Two deletions involved cut sites in inverted tetrameric repeats; one of these also involved an insertion.

The formation of mitochondrial deletions brought about by FS20 irradiation was examined in a line of human epithelial cells. In this system, Mt DNA deletions were found to be induced within 24 h following a single irradiation as low as 1.4 mJ/cm2 from an FS20 light source. We observed at least two distinct FS20-induced Mt deletions: the widely observed 4977 bp common deletion (CD) and a novel 5128 bp deletion flanked by TAGG repeats at nt 8247–8250 and 13,375–13,378 that has not, to our knowledge been previously described. While glutathione (GSH) at concentrations between 50 and 200 μM was found to block the formation of the CD in a dose-dependent manner, GSH at higher concentration induced the CD even in unirradiated cells. This is consistent with the idea that low dose GSH inhibits CD formation via reductive elimination of peroxides while higher concentrations may act as an electron donor to produce reactive oxygen species. Interestingly, in irradiated cells, GSH at intermediate concentrations (50 μM) induced a second, shorter, deletion similar to the novel deletion induced by FS20 alone but involving a different TAGG repeat spanning nt 13,175–13,178.

Background SIRT6, a member of the NAD+-dependent histone/protein deacetylase family, regulates genomic stability, metabolism, and lifespan. MYH glycosylase and APE1 are two base excision repair (BER) enzymes involved in mutation avoidance from oxidative DNA damage. Rad9–Rad1–Hus1 (9–1–1) checkpoint clamp promotes cell cycle checkpoint signaling and DNA repair. BER is coordinated with the checkpoint machinery and requires chromatin remodeling for efficient repair. SIRT6 is involved in DNA double-strand break repair and has been implicated in BER. Here we investigate the direct physical and functional interactions between SIRT6 and BER enzymes. Results We show that SIRT6 interacts with and stimulates MYH glycosylase and APE1. In addition, SIRT6 interacts with the 9-1-1 checkpoint clamp. These interactions are enhanced following oxidative stress. The interdomain connector of MYH is important for interactions with SIRT6, APE1, and 9–1–1. Mutagenesis studies indicate that SIRT6, APE1, and Hus1 bind overlapping but different sequence motifs on MYH. However, there is no competition of APE1, Hus1, or SIRT6 binding to MYH. Rather, one MYH partner enhances the association of the other two partners to MYH. Moreover, APE1 and Hus1 act together to stabilize the MYH/SIRT6 complex. Within human cells, MYH and SIRT6 are efficiently recruited to confined oxidative DNA damage sites within transcriptionally active chromatin, but not within repressive chromatin. In addition, Myh foci induced by oxidative stress and Sirt6 depletion are frequently localized on mouse telomeres. Conclusions Although SIRT6, APE1, and 9-1-1 bind to the interdomain connector of MYH, they do not compete for MYH association. Our findings indicate that SIRT6 forms a complex with MYH, APE1, and 9-1-1 to maintain genomic and telomeric integrity in mammalian cells. Electronic supplementary material The online version of this article (doi:10.1186/s12867-015-0041-9) contains supplementary material, which is available to authorized users.

In the supplement Material Section results from the protein purification and respective SDS-PAGE as well as the data from the stimulation of a SIRT6 mutant on MYH and APE1 activities are presented.

SIRT1, a member of the NAD(+)-dependent histone/protein deacetylase family, is involved in chromatin remodeling, DNA repair, and stress response and is a potential drug target. 5-fluorouracil (FU) and the SN1-type DNA methylating agent temozolomide (TMZ) are anticancer agents. In this study, we demonstrate that sirt1 knockout mouse embryonic fibroblast cells are more sensitive to FU and DNA methylating agents than normal cells. Based on these findings, the chemotherapy efficacy of SIRT1 inhibitors in combination with FU or TMZ were tested with human breast cancer cells. We found that treatments combining SIRT1 inhibitors with FU or TMZ show synergistic reduction of cell viability and colony formation of breast cancer cells. Thus, inhibition of SIRT1 activity provides a novel anticancer strategy.

Herpesviruses can establish a persistent infection in the cells and tissues of their natural hosts and thus may produce diseases due to cytolytic infections. We have isolated a herpesvirus from a bovine vascular endothelial cell culture after continuous subculturing. Typical cytopathic changes were observed in bovine endothelial cell cultures 2 days after inoculation of the virus. The virus had an icosahedral nucleocapsid of 100-150 nm in diameter and an envelope. The sequences of some DNA fragments of the virus were highly homologous to those of the bovine herpesvirus type 4 (BHV-4) strains. The DNA restriction maps of the virus and the reference strains of BHV-4, DN 599 and Movar 33/63 were very similar but not identical. Therefore, the newly isolated virus has been designated Taiwan strain. The presence of BHV-4 DNA in apparently normal bovine endothelial cell cultures was shown by Southern blot hybridization with the BamHI fragment of the newly isolated BHV-4 and was further confirmed by digestion of the DNA with BamHI plus AccI. In conclusion, we have demonstrated that BHV-4 persisted in the bovine endothelial cell cultures and continuous subcultures could lead to the production of infectious viral particles.

TDG (thymine DNA glycosylase) is an essential multifunctional enzyme involved in DNA base excision repair, DNA demethylation and transcription regulation. TDG is the predominant enzyme that removes thymine from T/G mispair, which arises due to deamination of 5-methyl-cytosine at the CpG dinucleotide, thereby preventing C to T mutations. SIRT1 is a member of class III NAD+-dependent histone/protein deacetylases. In the present study, we demonstrate that SIRT1 interacts with residues 67–110 of hTDG (human TDG). In addition, SIRT1 enhances TDG glycosylase activity and deacetylates acetylated TDG. TDG acetylation weakens its interaction with SIRT1. Although acetylated TDG has reduced glycosylase activity towards T/G, 5-formylcytosine/G and 5-carboxylcytosine/G, it has a stronger activity towards a 5-fluorouracil/G substrate as compared with unmodified TDG. SIRT1 weakly stimulates acetylated hTDG activity towards T/G, 5-formylcytosine/G and 5-carboxylcytosine/G as compared with control hTDG. Sirt1-knockout mouse embryonic fibroblast cells have higher levels of TDG expression and acetylation. The physical and functional interactions between SIRT1 and TDG may mediate DNA repair, gene expression and FU (5-fluorouracil)-mediated cytotoxicity.

Oxidative DNA damage is repaired primarily by the base excision repair (BER) pathway in a process initiated by removal of base lesions or mismatched bases by DNA glycosylases. MutY homolog (MYH, MUTYH, or Myh1) is a DNA glycosylase which excises adenine paired with the oxidative lesion 8-oxo-7,8-dihydroguanine (8-oxoG, or G°), thus reducing G:C to T:A mutations. The resulting apurinic/apyrimidinic (AP) site is processed by an AP-endonuclease or a bifunctional glycosylase/lyase. We show here that the major Schizosaccharomyces pombe AP endonuclease, Apn2, binds to the inter-domain connector located between the N- and C-terminal domains of Myh1. This Myh1 inter-domain connector also interacts with the Hus1 subunit of the Rad9-Rad1-Hus1 checkpoint clamp. Mutagenesis studies indicate that Apn2 and Hus1 bind overlapping but different sequence motifs on Myh1. Mutation on I(261) of Myh1 reduces its interaction with Hus1, but only slightly attenuates its interaction with Apn2. However, E(262) of Myh1 is a key determinant for both Apn2 and Hus1 interactions. Like human APE1, Apn2 has 3'-phosphodiesterase activity. However, unlike hAPE1, Apn2 has a weak AP endonuclease activity which cleaves the AP sites generated by Myh1 glycosylase. Functionally, Apn2 stimulates Myh1 glycosylase activity and Apn2 phosphodiesterase activity is stimulated by Myh1. The cross stimulation of Myh1 and Apn2 enzymatic activities is dependent on their physical interaction. Thus, Myh1 and Apn2 constitute an initial BER complex.

A number of factors have been identified that increase the risk of hepatocellular carcinoma (HCC). Recently it has become appreciated that type II diabetes increases the risk of developing HCC. This represents a patient population that can be identified and targeted for cancer prevention. The biguanide metformin is a first-line therapy for the treatment of type II diabetes in which it exerts its effects primarily on the liver. A role of metformin in HCC is suggested by studies linking metformin intake for control of diabetes with a reduced risk of HCC. Although a number of preclinical studies show the anticancer properties of metformin in a number of tissues, no studies have directly examined the effect of metformin on preventing carcinogenesis in the liver, one of its main sites of action. We show in these studies that metformin protected mice against chemically induced liver tumors. Interestingly, metformin did not increase AMPK activation, often shown to be a metformin target. Rather metformin decreased the expression of several lipogenic enzymes and lipogenesis. In addition, restoring lipogenic gene expression by ectopic expression of the lipogenic transcription factor SREBP1c rescues metformin-mediated growth inhibition. This mechanism of action suggests that metformin may also be useful for patients with other disorders associated with HCC in which increased lipid synthesis is observed. As a whole these studies show that metformin prevents HCC and that metformin should be evaluated as a preventive agent for HCC in readily identifiable at-risk patients. Cancer Prev Res; 5(4); 544–52. ©2012 AACR.

Despite the role of aerobic glycolysis in cancer, recent studies highlight the importance of the mitochondria and biosynthetic pathways as well. PPARγ coactivator 1α (PGC1α) is a key transcriptional regulator of several metabolic pathways including oxidative metabolism and lipogenesis. Initial studies suggested that PGC1α expression is reduced in tumors compared with adjacent normal tissue. Paradoxically, other studies show that PGC1α is associated with cancer cell proliferation. Therefore, the role of PGC1α in cancer and especially carcinogenesis is unclear. Using Pgc1α−/− and Pgc1α+/+ mice, we show that loss of PGC1α protects mice from azoxymethane-induced colon carcinogenesis. Similarly, diethylnitrosamine-induced liver carcinogenesis is reduced in Pgc1α−/− mice as compared with Pgc1α+/+ mice. Xenograft studies using gain and loss of PGC1α expression showed that PGC1α also promotes tumor growth. Interestingly, while PGC1α induced oxidative phosphorylation and tricarboxylic acid cycle gene expression, we also observed an increase in the expression of two genes required for de novo fatty acid synthesis, ACC and FASN. In addition, SLC25A1 and ACLY, which are required for the conversion of glucose into acetyl-CoA for fatty acid synthesis, were also increased by PGC1α, thus linking the oxidative and lipogenic functions of PGC1α. Indeed, using stable 13C isotope tracer analysis, we show that PGC1α increased de novo lipogenesis. Importantly, inhibition of fatty acid synthesis blunted these progrowth effects of PGC1α. In conclusion, these studies show for the first time that loss of PGC1α protects against carcinogenesis and that PGC1α coordinately regulates mitochondrial and fatty acid metabolism to promote tumor growth. Cancer Res; 71(21); 6888–98. ©2011 AACR.

Solar radiation can lead to changes affecting DNA metabolism resulting in loss of DNA integrity. Skin specimens obtained from melanoma patients treated at the Memorial Sloan-Kettering Cancer Center were used to study patterns of DNA fragmentation using the comet assay and levels of deletions in mitochondrial DNA (mtDNA) using real-time PCR. Skin specimens were classified according to the glutathione S-transferase M1 (GSTM1) genotype (either wild type [WT] or null) and patient sunburn history. GSTM1 null individuals with a sunburn history showed increased levels of both DNA fragmentation by comet assays and mtDNA deletions relative to GSTM1 WT patients with little or no sunburn history. Microarray analyses identified a number of genes whose expression was upregulatedor=5-fold in cells from GSTM1-null patients or from those reporting histories of sunburn. These genes encoded small molecule transporters, various growth factor/chemokine receptors, transcription factors and tumor suppressors. Of 17 genes directly involved in DNA repair, three DNA ligases were highly upregulated while the RAD23 UV excision repair gene and the Growth Arrest and DNA Damage gene (GADD45) were downregulated. These findings support the idea that exposure to solar radiation early in life may induce long-term cellular changes that lead to persistent DNA damage and altered patterns of gene expression.

A retrospective study of nucleotide sequence alterations in exons 7-9 of the E-cadherin gene and expression of E-cadherin and beta-catenin in gastric tumors from African American, Asian, Causcasian and Hispanic patients was carried out to determine differences potentially related to race/ethnicity in these groups.Paraffin-embedded tissue sections archived at the Memorial Sloan Kettering Cancer Center were used for immunohistochemical staining of sections for membranous E-cadherin protein and nuclear localization of beta-catenin. DNA from tumor tissue extracted from the paraffin sections was used as template for amplification of the E-cadherin gene exonic regions.Sequence analyses of the high-frequency mutation region along E-cadherin exons 7-9 revealed a number sequence alterations in the patient group as a whole, mostly within exon 8. The alterations were mainly single nucleotide insertions, but a palindromic duplication of exon 8 in a Caucasian patient and several extragenic insertions in a Hispanic and an African American patient were also found. Nuclear localization of beta-catenin was correlated with inactivating sequence alterations in three patients.Exon 8 was found to display the most extensive alterations in all the groups studied. As a group, the most extensive sequence alterations were found in tumors from Caucasian patients. A finding of potential significance as a biomarker related to ethnicity was a C insertion at nt 76,598 found independently in two African American patients.

Arsenic is a prevalent environmental carcinogen but arsenic is not directly mutagenic and the mechanism by which arsenite brings about oncogenic transformation is poorly understood. To gain insight into the oncogenic properties of arsenic, we studied the expression of cyclin D1 in cultured human epidermal keratinocytes treated with submicromolar concentrations of sodium arsenite. Arsenite at concentrations between 200 and 800 nM over a 3-day period brought about an increase in cell growth rate. Uptake of the vital stain, neutral red, arsenite at 200 and 400 nM concentrations brought about a parallel increase in cell viability over the same treatment period. Analysis of cell cycle parameters by flow cytometry showed that the growth stimulation was accompanied by a concomitant shift from the G1 into the S/G2 cell cycle compartment in the arsenite-treated cells. Real-time PCR analysis of cyclin D1 transcription showed that there was an induction of more than three-fold in cells exposed to 400 nM arsenite for 3 days. Quantitation of cyclin D levels in Western blots showed that arsenite treatment caused a time-dependent induction of cyclin D proteins representing an induction of about 2.0-fold after a 7 day treatment period. Electrophoretic mobility shift assays (EMSA) showed that arsenite also stimulated binding of the transcription factors, AP1 and CREBP to their respective binding motifs within 3 days. This supports a mechanism of oncogenesis based on persistent upregulation of D type cyclins leading to a concomitant loss of G1/S checkpoint control.

We compared the effects of overexpressing a tightly regulated anti-inflammatory cytokine, interleukin 10 (IL-10), and the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) on sulfur mustard induced cytotoxicity in human epidermal keratinocytes. Both cytokines were overexpressed when compared with the cells transfected with the empty vector as determined by quantitative ELISA. Cells overexpressing interleukin 10 suppressed the pro-inflammatory cytokines interleukin 8 and interleukin 6 following exposure to 50–300 μM sulfur mustard. These cells exhibited delayed onset of sulfur mustard induced cell death. On the other hand, cells overexpressing tumor necrosis factor alpha induced a sustained elevation in both interleukin 6 and 8 expression following exposure to 50–300 μM sulfur mustard. These cells were sensitized to the effects of sulfur mustard that resulted in an increased sulfur mustard induced cell death. Normal human epidermal keartinocytes treated with sulfur mustard exhibited elevated levels of tumor necrosis factor alpha expression and increased activity of nuclear factor kappa B. Gene array data indicated that cells overexpressing interleukin 10 induced several genes that are involved in growth promotion and cell-fate determination. We, therefore, identify IL-10 and TNF-α signal transduction pathways and their components as possible candidates for early therapeutic intervention against sulfur mustard induced cell injury. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:213–225, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20089