Defining the Role for ZBP-89 in ATM-mediated DNA Damage Response to Irradiation in the Intestine

ZBP-89 (ZNF148, Zfp148) is a zinc-finger transcription factor that inhibits cellular proliferation when overexpressed in cell lines. ZBP-89 forms a protein-protein interaction with p53 and Ataxia-telangiectasia mutated (ATM). However, it is unclear how the interaction modulates the function of these two proteins in vivo. Double strand DNA breakage induced by -irradiation induces ATM phosphoinositol kinase activity, which initiates a cascade of events culminating in cell cycle arrest, DNA repair or apoptosis. Elevated levels of ZBP-89 induce growth arrest and apoptosis in gastrointestinal cell lines. Therefore, we hypothesize that ZBP-89 facilitates cell growth arrest and activation of the DNA repair pathway after ATM activation. To test this hypothesis, we irradiated 4 groups of mice—1) C57BL/6 mice without any genetic deletion; 2) mice with one or both copies of ATM deleted from the intestinal mucosa; 3) mice with the ATM deletion and one copy of Zfp148 deleted; 4) ATM deletion with both copies of Zfp148 deleted. After the intestines were fixed, paraffin-embedded and sectioned, I performed the de-paraffinization step for immunohistochemistry of ZBP-89, p53 and H2AX. To stain for ZBP-89 protein, I performed an antigen retrieval step using sodium citrate, followed by blocking with 3% hydrogen peroxide and serum. I then added the primary antibody then the secondary antibodies. I used diaminobenzidine (DAB), a chromogenic detection method to visualize the antigen-antibody complex. I then counterstained with hematoxylin. We predict that the mice with a deletion of ZBP-89 will exhibit more tissue damage as a result of the irradiation and DNA damage. Results are pending

Secondary Prevention of Colorectal Cancer: Is There an Optimal Follow-up for Patients with Colorectal Cancer?

Secondary prevention of colorectal cancer, as opposed to primary prevention, indicates that a person has already had the disease and there are steps being taken to prevent cancer recurrence, usually as metachronous tumors. This generally involves annual surveillance with colonoscopy after surgical removal of the initial cancer if some aspect of the colon remains. However, some familial cases may involve other modalities, such as cyclooxygenase inhibitors, as an adjunct after the initial operation. Genetic testing in suspected familial cases may identify candidates for secondary prevention. The timing for secondary prevention is critical to prevent recurrent advanced disease, which is detrimental to patient survival. Recommendations are often empiric, but some cases are based on the biological behavior of the tumor. Close follow-up with a competent health care provider, such as a gastroenterologist, is necessary to help prevent recurrence

ArterioVenous Malformation within Jejunal Diverticulum: An Unusual Cause of Massive Gastrointestinal Bleeding

Massive gastrointestinal (GI) bleeding can occur with multiple jejunal diverticulosis. However, significant bleeding in the setting of few diverticulae is very unusual and rare. We report a case of massive gastrointestinal bleeding from an arteriovenous malformation (AVM) within a jejunal diverticulum to underscore the significance of such coexisting pathologies. Mesenteric angiogram was chosen to help identify the source of bleeding and to offer an intervention. Despite endovascular coiling, emergent intestinal resection of the bleeding jejunal segment was warranted to ensure definitive treatment. However several reports have shown jejunal diverticulosis as a rare cause of massive GI bleeding. The coexistence of jejunal diverticulum and AVM is rare and massive bleeding from an acquired Dieulafoy-like AVM within a diverticulum has never previously been described. Awareness of Dieulafoy-like AVM within jejunoileal diverticulosis is useful in preventing delay in treatment

Delayed Gastric Emptying after Laparoscopic Anterior Highly Selective and Posterior Truncal Vagotomy

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75042/1/j.1572-0241.1995.tb09325.x.pd

Clinical and Genetic Factors to Inform Reducing Colorectal Cancer Disparitites in African Americans

Colorectal cancer (CRC) is the third most prevalent and second deadliest cancer in the U.S. with 140,250 cases and 50,630 deaths for 2018. Prevention of CRC through screening is effective. Among categorized races in the U.S., African Americans (AAs) show the highest incidence and death rates per 100,000 when compared to Non-Hispanic Whites (NHWs), American Indian/Alaskan Natives, Hispanics, and Asian/Pacific Islanders, with an overall AA:NHW ratio of 1.13 for incidence and 1.32 for mortality (2010-2014, seer.cancer.gov). The disparity for CRC incidence and worsened mortality among AAs is likely multifactorial and includes environmental (e.g., diet and intestinal microbiome composition, prevalence of obesity, use of aspirin, alcohol, and tobacco use), societal (e.g., socioeconomic status, insurance and access to care, and screening uptake and behaviors), and genetic (e.g., somatic driver mutations, race-specific variants in genes, and inflammation and immunological factors). Some of these parameters have been investigated, and interventions that address specific parameters have proven to be effective in lowering the disparity. For instance, there is strong evidence raising screening utilization rates among AAs to that of NHWs reduces CRC incidence to that of NHWs. Reducing the age to commence CRC screening in AA patients may further address incidence disparity, due to the earlier age onset of CRC. Identified genetic and epigenetic changes such as reduced MLH1 hypermethylation frequency, presence of inflammation-associated microsatellite alterations, and unique driver gene mutations (FLCN and EPHA6) among AA CRCs will afford more precise approaches toward CRC care, including the use of 5-fluorouracil and anti-PD-1

Performance of multitarget stool DNA testing in African American patients

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146600/1/cncr31660_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146600/2/cncr31660.pd

Detection of Multiple Human Papillomavirus Genotypes in Anal Carcinoma

Infection with human papillomavirus (HPV) is a major risk factor for development of anal squamous cell carcinoma. Despite over 100 genotypes of the virus, HPV 16 and 18 are considered pathogenic as they are seen in the majority of cervical and anal cancers. We have employed a custom microarray to examine DNA for several HPV genotypes. We aimed to determine the accuracy of our microarray in anal cancer DNA for HPV genotypes compared to the DNA sequencing gold standard

Activin Signaling in Microsatellite Stable Colon Cancers Is Disrupted by a Combination of Genetic and Epigenetic Mechanisms

Activin receptor 2 (ACVR2) is commonly mutated in microsatellite unstable (MSI) colon cancers, leading to protein loss, signaling disruption, and larger tumors. Here, we examined activin signaling disruption in microsatellite stable (MSS) colon cancers.Fifty-one population-based MSS colon cancers were assessed for ACVR1, ACVR2 and pSMAD2 protein. Consensus mutation-prone portions of ACVR2 were sequenced in primary cancers and all exons in colon cancer cell lines. Loss of heterozygosity (LOH) was evaluated for ACVR2 and ACVR1, and ACVR2 promoter methylation by methylation-specific PCR and bisulfite sequencing and chromosomal instability (CIN) phenotype via fluorescent LOH analysis of 3 duplicate markers. ACVR2 promoter methylation and ACVR2 expression were assessed in colon cancer cell lines via qPCR and IP-Western blots. Re-expression of ACVR2 after demethylation with 5-aza-2'-deoxycytidine (5-Aza) was determined. An additional 26 MSS colon cancers were assessed for ACVR2 loss and its mechanism, and ACVR2 loss in all tested cancers correlated with clinicopathological criteria.Of 51 MSS colon tumors, 7 (14%) lost ACVR2, 2 (4%) ACVR1, and 5 (10%) pSMAD2 expression. No somatic ACVR2 mutations were detected. Loss of ACVR2 expression was associated with LOH at ACVR2 (p<0.001) and ACVR2 promoter hypermethylation (p<0.05). ACVR2 LOH, but not promoter hypermethylation, correlated with CIN status. In colon cancer cell lines with fully methylated ACVR2 promoter, loss of ACVR2 mRNA and protein expression was restored with 5-Aza treatment. Loss of ACVR2 was associated with an increase in primary colon cancer volume (p<0.05).Only a small percentage of MSS colon cancers lose expression of activin signaling members. ACVR2 loss occurs through LOH and ACVR2 promoter hypermethylation, revealing distinct mechanisms for ACVR2 inactivation in both MSI and MSS subtypes of colon cancer

5,10-Methylenetetrahydrofolate Reductase 677 and 1298 Polymorphisms, Folate Intake, and Microsatellite Instability in Colon Cancer

The 5,10-methylenetetrahydrofolate reductase (MTHFR) gene plays a critical role in folate metabolism. Studies on the association between MTHFR polymorphisms and length changes in short tandem repeat DNA sequences [microsatellite instability (MSI)] are inconsistent. Using data from colon cancer cases (n = 503) enrolled as part of an existing population-based case-control study, we investigated the association between MTHFR 677 and MTHFR 1298 polymorphisms and MSI. We also examined whether the association was modified by folate intake. Participants were case subjects enrolled as part of the North Carolina Colon Cancer Study. Consenting cases provided information about lifestyle and diet during in-home interviews as well as blood specimens and permission to obtain tumor blocks. DNA from normal and tumor tissue sections was used to determine microsatellite status (MSI). Tumors were classified as MSI if two or more microsatellite markers (BAT25, BAT26, D5S346, D2S123, and D17S250) had changes in the number of DNA sequence repeats compared with matched nontumor tissue. Tumors with one positive marker (MSI-low) or no positive markers (microsatellite stable) were grouped together as non-MSI tumors (microsatellite stable). MTHFR 677 and MTHFR 1298 genotypes were determined by real-time PCR using the 5′ exonuclease (Taqman) assay. Logistic regression was used to calculate odds ratio (OR) and 95% confidence intervals (95% CI). MSI was more common in proximal tumors (OR, 3.8; 95% CI, 1.7–8.4) and in current smokers (OR, 4.0; 95% CI, 1.6–9.7). Compared with MTHFR 677 CC referent, MTHFR 677 CT/TT genotype was inversely associated with MSI among White cases (OR, 0.36; 95% CI, 0.16–0.81) but not significant among African Americans. Although not statistically significant, a similar inverse association was observed between MTHFR 677 CT/TT genotype and MSI among the entire case subjects (OR, 0.54; 95% CI, 0.26–1.10). Among those with adequate folate intake (>400 μg total folate), we found strong inverse associations between combined MTHFR genotypes and MSI (677 CC + 1298 AC/CC, OR, 0.09; 95% CI, 0.01–0.59; 677 CT/TT + 1298 AA, OR, 0.13; 95% CI, 0.02–0.85) compared with the combined wild-type genotypes (677 CC + 1298 AA). This protective effect was not evident among those with low folate (<400 μg total folate) intake. Our results suggest that MTHFR variant genotypes are associated with reduced risk of MSI tumors under conditions of adequate folate intake, although the data are imprecise due to small numbers. These results indicate that the relationship between MTHFR genotypes and MSI is influenced by folate status

Mutation Rates of TGFBR2 and ACVR2 Coding Microsatellites in Human Cells with Defective DNA Mismatch Repair

Microsatellite instability promotes colonic tumorigenesis through generating frameshift mutations at coding microsatellites of tumor suppressor genes, such as TGFBR2 and ACVR2. As a consequence, signaling through these TGFβ family receptors is abrogated in DNA Mismatch repair (MMR)-deficient tumors. How these mutations occur in real time and mutational rates of these human coding sequences have not previously been studied. We utilized cell lines with different MMR deficiencies (hMLH1−/−, hMSH6−/−, hMSH3−/−, and MMR-proficient) to determine mutation rates. Plasmids were constructed in which exon 3 of TGFBR2 and exon 10 of ACVR2 were cloned +1 bp out of frame, immediately after the translation initiation codon of an enhanced GFP (EGFP) gene, allowing a −1 bp frameshift mutation to drive EGFP expression. Mutation-resistant plasmids were constructed by interrupting the coding microsatellite sequences, preventing frameshift mutation. Stable cell lines were established containing portions of TGFBR2 and ACVR2, and nonfluorescent cells were sorted, cultured for 7–35 days, and harvested for flow cytometric mutation detection and DNA sequencing at specific time points. DNA sequencing revealed a −1 bp frameshift mutation (A9 in TGFBR2 and A7 in ACVR2) in the fluorescent cells. Two distinct fluorescent populations, M1 (dim, representing heteroduplexes) and M2 (bright, representing full mutants) were identified, with the M2 fraction accumulating over time. hMLH1 deficiency revealed 11 (5.91×10−4) and 15 (2.18×10−4) times higher mutation rates for the TGFBR2 and ACVR2 microsatellites compared to hMSH6 deficiency, respectively. The mutation rate of the TGFBR2 microsatellite was ∼3 times higher in both hMLH1 and hMSH6 deficiencies than the ACVR2 microsatellite. The −1 bp frameshift mutation rates of TGFBR2 and ACVR2 microsatellite sequences are dependent upon the human MMR background