Association of miR-146a-5p and miR-21-5p with Prognostic Features in Melanomas.

BACKGROUND: Cutaneous melanoma (CM) is one of the most lethal tumors among skin cancers and its incidence is rising worldwide. Recent data support the role of microRNAs (miRNAs) in melanoma carcinogenesis and their potential use as disease biomarkers. METHODS: We quantified the expression of miR-146a-5p and miR-21-5p in 170 formalin-fixed paraffin embedded (FFPE) samples of CM, namely 116 superficial spreading melanoma (SSM), 26 nodular melanoma (NM), and 28 lentigo maligna melanoma (LMM). We correlated miRNA expression with specific histopathologic features including Breslow thickness (BT), histological subtype, ulceration and regression status, and mitotic index. RESULTS: miR-146a-5p and miR-21-5p were significantly higher in NM compared to SSM and LMM. The positive correlation between miR-146a-5p and miR-21-5p expression and BT was confirmed for both miRNAs in SSM. Considering the ulceration status, we assessed that individual miR-21-5p expression was significantly higher in ulcerated CMs. The increased combined expression of the two miRNAs was strongly associated with ulceration (p = 0.0093) and higher mitotic rate (≥1/mm2) (p = 0.0005). We demonstrated that the combination of two-miRNA expression and prognostic features (BT and ulceration) can better differentiate cutaneous melanoma prognostic groups, considering overall survival and time-to-relapse clinical outcomes. Specifically, miRNA expression can further stratify prognostic groups among patients with BT ≥ 0.8 mm but without ulceration. Our findings provide further insights into the characterization of CM with specific prognostic features. The graphical abstract was created with BioRender.com.

Wilson disease, ABCC2 c.3972C > T polymorphism and primary liver cancers: suggestions from a familial cluster.

BACKGROUND: Polymorphisms in genes modulating xenobiotics metabolism, in particular the ABCC2 c.3972C > T single nucleotide polymorphism (SNP) at exon 28, have been suggested to increase primary liver cancer (PLC) risk. Conversely, the occurrence of PLCs in Wilson disease patients is a rare event, in contrast with the occurrence observed in other chronic liver diseases. Here we report the clinical case of five siblings carrying the ABCC2 c.3972C > T SNP; three of them were affected by Wilson disease and two brothers with Wilson disease also developed PLCs. METHODS: The presence of the ABCC2 c.3972C > T SNP was assessed by Sanger sequencing and the exposure of PLC risk factors by standardized questionnaires. RESULTS: Notably, PLCs occurred only in the two brothers with the ABCC2 c.3972C > T SNP and Wilson disease who resulted exposed to asbestos and cigarette smoking, but not in the other siblings with the ABCC2 c.3972C > T SNP, alone or in association with Wilson disease, not exposed to these carcinogens and/or to other known risk factors for PLCs. CONCLUSIONS: These findings suggest that ABCC2 c.3972C > T SNP and WD, also in association, may not represent a sufficient condition for PLC development, but that co-occurrence of further host/exogenous risk factors are needed to drive this process, reinforcing the notion that liver carcinogenesis is the result of a complex interplay between environmental and host genetic determinants. Due to the sporadic cases of this study and the paucity of data currently available in literature on this issue, future investigations in a larger population are needed to confirm our findings.

Gene Panel Analysis in a Large Cohort of Patients With Autosomal Dominant Polycystic Kidney Disease Allows the Identification of 80 Potentially Causative Novel Variants and the Characterization of a Complex Genetic Architecture in a Subset of Families.

Introduction: Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited disorders in humans and the majority of patients carry a variant in either PKD1 or PKD2. Genetic testing is increasingly required for diagnosis, prognosis, and treatment decision, but it is challenging due to segmental duplications of PKD1, genetic and allelic heterogeneity, and the presence of many variants hypomorphic or of uncertain significance. We propose an NGS-based testing strategy for molecular analysis of ADPKD and its phenocopies, validated in a diagnostic setting. Materials and Methods: Our protocol is based on high-throughput simultaneous sequencing of PKD1 and PKD2 after long range PCR of coding regions, followed by a masked reference genome alignment, and MLPA analysis. A further screening of additional 14 cystogenes was performed in negative cases. We applied this strategy to analyze 212 patients with a clinical suspicion of ADPKD. Results and Discussion: We detected causative variants (interpreted as pathogenic/likely pathogenic) in 61.3% of our index patients, and variants of uncertain clinical significance in 12.5%. The majority (88%) of genetic variants was identified in PKD1, 12% in PKD2. Among 158 distinct variants, 80 (50.6%) were previously unreported, confirming broad allelic heterogeneity. Eleven patients showed more than one variant. Segregation analysis indicated biallelic disease in five patients, digenic in one, de novo variant with unknown phase in two. Furthermore, our NGS protocol allowed the identification of two patients with somatic mosaicism, which was undetectable with Sanger sequencing. Among patients without PKD1/PKD2 variants, we identified three with possible alternative diagnosis: a patient with biallelic mutations in PKHD1, confirming the overlap between recessive and dominant PKD, and two patients with variants in ALG8 and PRKCSH, respectively. Genotype-phenotype correlations showed that patients with PKD1 variants predicted to truncate (T) the protein experienced end-stage renal disease 9 years earlier than patients with PKD1 non-truncating (NT) mutations and >13 years earlier than patients with PKD2 mutations. ADPKD-PKD1 T cases showed a disease onset significantly earlier than ADPKD-PKD1 NT and ADPK-PKD2, as well as a significant earlier diagnosis. These data emphasize the need to combine clinical information with genetic data to achieve useful prognostic predictions.

Partial Portal Vein Arterialization to Treat Toxic Acute Liver Failure in Elderly Rats.

INTRODUCTION: Toxic acute liver failure can be fatal even after liver transplantation. Since there are data only in young rats, the aim of our study was to verify the effectiveness of the increase of oxygen to the liver by partial portal vein arterialization (PPVA) in elderly rats with acute liver failure induced by carbon tetrachloride (CCl4) intoxication. METHODS: Twenty elderly (30 months) Sprague-Dawley rats underwent a CCl4 intoxication (5 mL/kg). Animals were divided after 24 hours (n = 10 per group) to either undergo PPVA (G1, treated group) or be untreated (G2, control group). PPVA consisted of a shunt between the left renal artery and splenic vein after nephrectomy and spelnectomy. The G2 group animals underwent nephrectomy and splenectomy only. The 10-day survival was evaluated. Before euthanasia, blood samples from the portal vein were detected for blood gas analysis. Liver injury was evaluated by the serum alanine aminotransferase (ALT) and prothrombin time levels. Histology was done to evaluate the liver necrosis. Hepatocyte regeneration was assessed by the mitotic index at immunohistochemistry. RESULTS: The PPVA has resulted in a significant increase in the oxygen partial pressure and saturation in portal blood. A survival improvement at 10 days was registered in the PPVA-treated rats (90% vs 30%; P = .0065). After 24 hours from intoxication, ALT was high in both groups. A rapid decrease of ALT was observed in G1 as compared to G2. At the same time, livers showed a severe centrolobular necrosis. In the suviving G2 rats, a moderate necrosis was present, while only a mild necrosis was observed in the G1 rats. An higher mitotic index was detected in rats treated with PPVA. CONCLUSIONS: In our experimental study, the presence of oxygenated blood in the portal venous system following the PPVA procedure had positive effects on liver regeneration and rats' survival. The PPVA treatment had beneficial effects in elderly rats.

A Mediterranean Diet Mix Has Chemopreventive Effects in a Murine Model of Colorectal Cancer Modulating Apoptosis and the Gut Microbiota.

Objectives: Unhealthy dietary patterns have been associated with colorectal cancer (CRC) onset while Mediterranean Diet (MD) has been proposed for CRC prevention. This study evaluated the effect of a Mediterranean Diet Mix (MD-MIX) on colonic tumors development in A/J mice fed a low-fat (LFD) or a high-fat western diet (HFWD), and injected with the procarcinogen azoxymethane (AOM). Materials and Methods: Forty A/J male mice were randomly assigned into four feeding arms (10 mice/arm; LFD, LFD-MD-MIX, HFWD, HFWD-MD-MIX) to be treated with AOM. Ten mice were exposed to the diets alone (Healthy LFD and Healthy HFWD) to be used as control. Tumor incidence and multiplicity were evaluated at sacrifice. Mucosal fatty acid content and urinary phenolic compounds were assayed by mass spectrometry. Apoptosis was evaluated by TUNEL assay and gene expression markers. Cell proliferation was evaluated by Ki67 immunohistochemistry. Microbiota composition was assessed at different time points by 16S RNA sequencing. Results: A tumor incidence of 100% was obtained in AOM-treated mice. The MD-MIX supplementation was able to reduce the number of colonic lesions in both LFD and HFWD-fed mice and to induce apoptosis, in particular in the LFD-MD-MIX arm. Moreover, a preventive effect on low-grade dysplasia and macroscopical lesions (>1 mm) development was found in HFWD-fed mice together with a regulation of the AOM-driven intestinal dysbiosis. Conclusions: MD-MIX was able to counteract CRC development in mice under different dietary backgrounds through the regulation of apoptosis and gut microbiota.

Short-term treatment with eicosapentaenoic acid improves inflammation and affects colonic differentiation markers and microbiota in patients with ulcerative colitis.

Patients with long-standing ulcerative colitis (UC) have an increased colorectal cancer (CRC) risk. In this pilot study we evaluated the effect of Eicosapentaenoic acid as free fatty acid (EPA-FFA) supplementation on mucosal disease activity, colonic differentiation markers and microbiota composition in UC patients. Twenty long-standing UC patients in stable clinical remission and with fecal calprotectin (FC) > 150 µg/g were enrolled (T0) and supplemented with EPA-FFA 2 g/daily for 90 days (T3). Endoscopic and histologic disease activities were measured by Mayo and Geboes scores, respectively. HES1, KLF4, STAT3, IL-10 and SOCS3 levels were determined using western blotting and qRT-PCR, while phospho-STAT3 levels were assessed by western blotting. Goblet cells were stained by Alcian blue. Microbiota analyses were performed on both fecal and colonic samples. Nineteen patients completed the study; seventeen (89.5%) were compliant. EPA-FFA treatment reduced FC levels at T3. Patients with FC > 150 µg/g at T3 (n = 2) were assumed as non-responders. EPA-FFA improved endoscopic and histological inflammation and induced IL-10, SOCS3, HES1 and KLF4 in compliant and responder patients. Importantly, long-term UC-driven microbiota composition was partially redressed by EPA-FFA. In conclusion, EPA-FFA supplementation reduced mucosal inflammation, promoted goblet cells differentiation and modulated intestinal microbiota composition in long-standing UC patients.

Anti-müllerian hormone and insulin-like 3 levels in healthy normal-weight ovulatory and anovulatory eumenorrheic late adolescent females: potential early biomarkers of ovarian dysfunction?

OBJECTIVE: The aim of this study was to evaluate differences in anti-müllerian hormone (AMH) and insulin-like 3 (INSL3) levels and their association with gonadotropin and ovarian steroid hormones, as expression of ovarian function, between healthy normal-weight ovulatory and anovulatory eumenorrheic late adolescent females. STUDY DESIGN: This study analyzed AMH and INSL3 levels in forty healthy eumenorrheic late adolescent females (aged 16-19 ys), selected from a cross-sectional epidemiological study performed on the prevalence of hyperandrogenic states. The subjects were divided into ovulatory (n: 28) and anovulatory (n: 12) groups in accordance to a previous cluster analysis based on progesterone (P) distribution measured once in the latter part of the cycle. Both groups were compared for anthropometric, biochemical and hormonal parameters. RESULTS: INSL3 and AMH were detectable in all samples. Testosterone (P=0.01), the free-androgen index (FAI) (P=0.051), gonadotropins (LH: P=0.02; FSH: P=0.004) and AMH (P=0.02) levels were significantly higher in the anovulatory group with respect to their ovulatory counterpart. A trend toward significantly higher INSL3 concentrations (P=0.08) was also shown in the anovulatory group. A positive correlation between INSL3 levels and androgens such as androstenedione (r=0.38; P=0.02), testosterone (r=0.44; P=0.004) and FAI (r=0.42; P=0.006) and a negative borderline significant correlation (r=-0.30; P=0.055) between AMH and P were shown in all subjects. CONCLUSION: Healthy eumenorrheic late adolescent females with sporadic anovulation display higher AMH and INSL-3 blood concentrations in association with higher androgen levels compared with age- and BMI-matched subjects with ovulatory cycle, suggesting evidence of an earlier ovarian dysfunction.

Portal blood arterialization with an extracorporeal device to treat toxic acute hepatic failure in a swine model.

PURPOSE: This study aimed to determine whether a controlled portal blood arterialization by a liver extracorporeal device (L.E.O2 NARDO) is effective in treating acute hepatic failure (AHF) induced through CCl4 administration in a swine model. METHODS: 20 swine with AHF induced by intraperitoneal injection of carbon tetrachloride (CCl4) in oil solution, were randomly divided into two groups: animals receiving L.E.O2 NARDO treatment 48 h after the intoxication (study group); animals sham operated 48 h after the intoxication (control group). Blood was withdrawn from the iliac artery and reversed in the portal venous system by an interposed extracorporeal device. Each treatment lasted 6 h. The survival was assessed at 5 days after L.E.O2 NARDO treatment or sham operation. In both groups blood samples were collected for biochemical analysis at different time points and liver biopsies were collected 48 h after intoxication and at sacrifice. RESULTS: We observed decreased transaminases levels and a more rapid INR recovery in the study group, as compared to the control group. Eight animals of the study group vs. two animals of the control group survived at five days after surgery with a statistically significant difference (p<0.05). Liver biopsies performed at sacrifice showed a reduction of the damaged hepatic areas in the study group as compared to the control group. CONCLUSIONS: Arterial blood supply in the portal system through the L.E.O2 NARDO device is easily applicable, efficacious, and safe in a swine model of AHF induced by CCl4 intoxication.

Parallel variations of insulin-like peptide 3 (INSL3) and antimüllerian hormone (AMH) in women with the polycystic ovary syndrome according to menstrual cycle pattern.

CONTEXT: Antimüllerian hormone (AMH) and insulin-like factor 3 (INSL3) represent ovarian functional markers of granulosa and theca cells, respectively. OBJECTIVE: We conducted a prospective study to investigate AMH and INSL3 plasma levels in 3 groups of women with polycystic ovary syndrome (PCOS) classified according to menstrual cyclicity pattern and their relationship with ovarian morphology and hormonal levels. DESIGN AND PARTICIPANTS: AMH and INSL3 were measured in a cohort of 57 patients with PCOS, divided into 3 groups according to menstrual status: eumenorrheic (PCOS-E, n = 15), oligomenorrheic (PCOS-O, n = 25), and amenorrheic (PCOS-A, n = 17). Clinical and endocrine characteristics and ovarian morphology were compared among the groups. Twenty-seven age- and weight-matched women without hyperandrogenism were included as controls. RESULTS: According to the menstrual pattern, the women with PCOS-A and PCOS-O had higher INSL3 levels with respect to the control women (P = .025 and P = .004, respectively) and higher but not significant INSL3 levels compared with those of the women with PCOS-E. AMH levels were significantly higher in women with PCOS-A and PCOS-O with respect to those in women with PCOS-E (P < .001 and P < .001, respectively) and control women (P < .001 and P < .001, respectively). Interestingly, a significant positive correlation was found between INSL3 and AMH blood levels in all women with PCOS (R = 0.43; P = .002) and across the groups (R = 0.41; P < .001). CONCLUSIONS: INSL3 and AMH levels are significantly correlated with each other in women with PCOS, and they are significantly increased, particularly in the presence of amenorrhea and oligomenorrhea. INSL3 and AMH may reflect a dysfunction of PCOS thecal and granulosa cells, which are responsible for the increased androgen production and chronic anovulation of this condition.

Effect of colic vein ligature in rats with loperamide-induced constipation.

INTRODUCTION: Medical treatment in chronic constipation is not always successful. Surgery is indicated in unresponsive selected severe cases. This study presents the distal venous colic ligation in rat as a novel surgical approach. MATERIALS AND METHODS: 16 rats (study group) were evaluated in 3 phases of 6 days each: A (normal conditions), B (loperamide-induced constipation), and C (colic vein legation) and compared with rats treated in phase C with PEG 4,000 (control group). Blood biochemical and physiological parameters, daily fecal water content (FWC), and histological analysis were performed in all study phases. RESULTS: No biochemical and physiological parameters changes were observed. FWC decreased in phase B and increased in phase C in both groups with a grow up to 2.3-fold in study group compared to control (P < 0.0001). Moreover, in study group, a high number of colonic goblet cells were detected (phase C versus phase B: P < 0.001) while no differences were registered in control. CONCLUSION: By ligature of the colic vein in constipated rats, an increase in FWC and goblet cells higher than in PEG treated rats was detected. The described surgical procedure appeared effective, simple, and safe; further studies in animal models, however, are necessary to assess its clinical applicability.

A new model for portal protein profile analysis in course of ileal intraluminal bile acid infusion using an in situ perfused rat intestine.

Due to the importance of intestinal transport in pharmacological studies and the emerging role of intestinal signaling activity in the gut-liver axis, we have developed a new method to investigate intestinal transport and liver signaling using cell and serum free mesenteric perfusion system in the rat. The method regarding bile acid active absorption was validated, then, the portal venous content was examined for fibroblast growth factor 15(FGF15), a putative signaling protein produced by the ileal enterocytes following bile acid absorption. After isolation and cannulation of the relevant vessels (abdominal aorta and portal vein), the abdominal aorta and the terminal ileum were infused with respectively Krebs-Ringer solution and tauroursodeoxycholate (TUDCA) and the absorption was assessed by its recovery in the portal vein. After immunoblot, liquid chromatography and mass spectrometry analysis were performed both on gel bands digestion products and on portal outflow samples in order to evaluate if negligible amounts of FGF15 were present in the portal circulation. TUDCA absorption was efficient, intestinal morphology and oxygen consumption were normal. Despite accurate analysis, we could not find FGF15. Our method proved to be reliable for studying the active bile acid absorption. It is also suitable to identify molecules produced by enterocytes and transferred to the portal circulation in response to absorption of different substances such as nutrients or drugs. Since FGF15 was not recovered we suggest the possibilities that this protein is produced in very little amounts, poorly transferred outside the cell, or that it is extremely unstable and rapidly degraded.

Doxorubicin coupled to lactosaminated albumin: effect of heterogeneity in drug load on conjugate disposition and hepatocellular carcinoma uptake in rats.

Coupling to lactosaminated human albumin (L-HSA) makes doxorubicin (DOXO) an effective drug against chemically induced rat hepatocellular carcinomas (HCCs). In the conjugate there is a large heterogeneity in the number of DOXO molecules bound to one L-HSA molecule. After lyophilization, the molecules with the higher DOXO load form large complexes (C-DOXOL), whereas those with low drug load (C-DOXOS) have the size of the carrier L-HSA. In the present experiments, we demonstrated that in C-DOXOL the molecules are not linked by covalent bonds, but are strongly aggregated probably because of mutual drug-drug interaction between the DOXO residues. In healthy rats and in animals with HCCs which received the same dose (1 microg/g) of DOXO injected in C-DOXOL or in C-DOXOS forms, penetration of the drug in tumors and in tissues was more rapid after administration of the former complex. Three hours after injection of both conjugate forms the intracellular release of DOXO from the carrier was completed. The AUCs from 0.5 to 4h of the levels of the released DOXO in HCCs, surrounding liver and bone marrow of animals injected with C-DOXOL were similar to those calculated in animals given C-DOXOS. This suggests that after administration of the dose of DOXO used in the present experiments the conjugate molecules with lower or higher drug load can exert comparable pharmacological and toxic effects.

A conjugate of doxorubicin with lactosaminated albumin enhances the drug concentrations in all the forms of rat hepatocellular carcinomas independently of their differentiation grade.

BACKGROUND/AIMS: Doxorubicin (DOXO) was coupled to lactosaminated human serum albumin (L-HSA) in order to enhance the drug concentration in the well differentiated hepatocellular carcinomas (HCCs), which can accumulate L-HSA through the asialoglycoprotein receptor. In the present experiments we compared the DOXO concentrations produced by this conjugate (L-HSA-DOXO) and by the uncoupled drug in the well, moderately, and poorly differentiated rat HCCs. METHODS: The same dose (1 microg/g) of free or L-HSA coupled-DOXO was injected in rats with HCCs induced by diethylnitrosamine. At different times, the animals were killed and the neoplastic nodules of liver were isolated. Their differentiation grade was determined histologically and their DOXO content was measured. RESULTS: Unexpectedly, we found that also in the poorly differentiated forms of HCCs, which display no or only a poor capacity of accumulating L-HSA, the conjugate raised DOXO levels that were approximately twofold higher than those produced by the free drug. CONCLUSIONS: The conjugate L-HSA-DOXO could improve the potential of DOXO in the treatment of all HCCs, including the poorly differentiated tumors that are the common forms in the advanced disease for which an effective chemotherapy is particularly needed.

Successful treatment of CCl4-induced acute liver failure with portal vein arterialization in the rat.

BACKGROUND: Optimization of the conditions for regeneration is a major goal in the management of patients with acute liver failure (ALF). Previous observations suggested that hyperoxygenation of the liver may improve its regenerative capacity. Thus, this study aimed to determine whether an additional supply of oxygenated blood achieved by portal vein arterialization (PVA) is protective in rat ALF caused by toxin administration or hepatectomy. METHODS: Sprague-Dawley rats were subjected or not to PVA after CCl(4) intoxication or extended hepatectomy. PVA was performed by interposing a stent between the left renal artery and splenic vein after left nephrectomy and splenectomy. Liver injury was evaluated by the serum ALT level and necrotic cell count. Hepatocyte regeneration was assessed by calculating the mitotic index and bromodeoxyuridine (BrdU) staining. The 10-day survival was assessed in separate experimental groups. RESULTS: The pO(2) in portal blood increased significantly following PVA. In the CCl(4)-induced ALF, serum ALT levels and necrosis were significantly reduced in arterialized than non-arterialized rats. PVA greatly promotes liver regeneration in both models. Finally, PVA significantly improved survival compared to controls (CCl(4): 100 versus 40%; 90% hepatectomy: 90 versus 30%). Interestingly, in the CCl(4)-induced ALF, survival was 100% even when the shunt was closed after 48 h. CONCLUSION: These data indicate that the additional supply of arterial oxygenated blood through PVA promotes a rapid regeneration leading to the resolution of toxic-induced massive liver necrosis and a faster restoration of liver mass after partial hepatectomy in rats. Thus, PVA may represent a novel tool for optimizing hepatocyte regeneration.

Enhanced uptake of lactosaminated human albumin by rat hepatocarcinomas: implications for an improved chemotherapy of primary liver tumors.

BACKGROUND/AIMS: The hepatocyte receptor for asialoglycoproteins (ASGP-R) internalizes macromolecules exposing galactosyl residues (MEGRs) which can be used as liver-addressed drug carriers. This receptor was also found on the cells of the large majority of well differentiated hepatocarcinomas (HCCs). The aim of the present experiments was to ascertain whether ASGP-R of HCCs is functionally active and these tumors can internalize higher quantities of MEGRs than extra-hepatic tissues. METHODS: We injected radioactive lactosaminated human albumin (L-HSA) in rats with HCCs produced by nitroso-diethylamine and measured the radioactivity of tumors, surrounding liver, heart, intestine and kidney. L-HSA is a MEGR successfully used in humans as a hepatotropic drug carrier. RESULTS: The levels of radioactivity of HCCs were two to three times lower than those of surrounding liver, but several times higher than those of extra-hepatic tissues. L-HSA accumulation in the tumors mainly occurred via the ASGP-R, as indicated by the 20 times lower penetration of non-lactosaminated HSA. L-HSA uptake by the well-differentiated tumors were four times higher compared with that by the poorly differentiated forms. CONCLUSIONS: The present results suggest that in the chemotherapy of HCCs expressing the ASGP-R the extra-hepatic toxicity of anticancer agents can be reduced by conjugation to L-HSA.

High thymidylate synthase expression in colorectal cancer with microsatellite instability: implications for chemotherapeutic strategies.

UNLABELLED: Colon cancers displaying microsatellite instability (MSI) are clinically less aggressive. Based on in vitro studies and recent clinical data, cancers displaying MSI do not respond to 5-fluorouracil (5-FU). The reasons why MSI tumors are clinically less aggressive and do not respond to 5-FU-based therapies have not been fully elucidated. PURPOSE: We investigated biomolecular markers in an attempt to explain the different clinical behavior and chemotherapeutic responses of MSI and non-MSI colon cancers. EXPERIMENTAL DESIGN: One hundred ninety-two sporadic colon cancers were tested for MSI with five mononucleotide markers and methylation of the hMLH1 promoter. Slides were stained for thymidylate synthase (TS), p53, MDM2, p21(WAF1/CIP1), beta-catenin, vascular endothelial growth factor, hMLH1, hMSH2, and hMSH6. Tumors were regarded as having wild-type, functional p53 (Fp53) if reduced expression of p53 and positive MDM2 and p21(WAF1/CIP1) expressions were found. RESULTS: Of the cases, 12.5% were MSI-H (at least two markers mutated). Of MSI-H cases, 83.3% were characterized by a complete loss of at least one of the mismatch repair proteins, in particular loss of hMLH1 by promoter hypermethylation. MSI-H colon cancers showed higher expression of TS compared with MSS (no mutated markers)/MSI-L (one mutated marker) colon cancers (66.6% for MSI-H versus 14.8% MSS/MSI-L; P < 0.0001); 20.8% of MSI-H cases showed high expression of the vascular endothelial growth factor, compared with 45.8% MSS/MSI-L colon cancers (P = 0.0005); 45.8% MSI-H cases had Fp53 compared 11.9% MSS/MSI-L cases (P < 0.0001). CONCLUSIONS: About 12% of colon cancers display MSI mostly due to lack of hMLH1 resulting from promoter hypermethylation. These tumors have high expression of TS and retain fully functional p53 system. Thus, these data suggest why sporadic hMLH1-defective colon cancers often do not respond to 5-FU.

Gene expression profiling in conjunction with physiological rescues of IKKalpha-null cells with wild type or mutant IKKalpha reveals distinct classes of IKKalpha/NF-kappaB-dependent genes.

Cellular responses to stress-like stimuli require the IkappaB kinase (IKK) signalsome (IKKalpha, IKKbeta, and NEMO/IKKgamma) to activate NF-kappaB-dependent genes. IKKbeta and NEMO/IKKgamma are required to release NF-kappaB p65/p50 heterodimers from IkappaBalpha, resulting in their nuclear migration and sequence-specific DNA binding; but IKKalpha was found to be dispensable for this initial phase of canonical NF-kappaB activation. Nevertheless, IKKalpha-/- mouse embryonic fibroblasts (MEFs) fail to express NF-kappaB targets in response to proinflammatory stimuli, uncovering a nuclear role for IKKalpha in NF-kappaB activation. However, it remains unknown whether the global defect in NF-kappaB-dependent gene expression of IKKalpha-/- cells is caused by the absence of IKKalpha kinase activity. We show by gene expression profiling that rescue of near physiological levels of wild type IKKalpha in IKKalpha-/- MEFs globally restores expression of their canonical NF-kappaB target genes. To prove that the kinase activity of IKKalpha was required on a genomic scale, the same physiological rescue was performed with a kinase-dead, ATP binding domain IKKalpha mutant (IKKalpha(K44M)). Remarkably, the IKKalpha(K44M) protein rescued approximately 28% of these genes, albeit in a largely stimulus-independent manner with the notable exception of several genes that also acquired tumor necrosis factor-alpha responsiveness. Thus the IKKalpha-containing signalsome unexpectedly functions in the presence and absence of extracellular signals in both kinase-dependent and -independent modes to differentially modulate the expression of five distinct classes of IKKalpha/NF-kappaB-dependent genes.

Induction of chromosomal instability in colonic cells by the human polyomavirus JC virus.

Most colorectal cancers display chromosomal instability, which is characterized by gross chromosomal rearrangements, loss of heterozygosity and aneuploidy. We have previously demonstrated a link between JC virus strains Mad-1 and Delta98 and colorectal cancer. Others have also associated the virus to the induction of colon cancer and aneuploid brain tumors by producing a highly tumorigenic protein named T antigen (TAg), which binds to beta-catenin and inactivates key proteins such as p53. The aim is to demonstrate that JC virus is capable of inducing chromosomal instability in colonic cells. We used the human colon cancer cell line RKO as a model. The cell line has wild-type p53, wild-type beta-catenin and APC and is diploid. Neuroblastoma JCI cells, which are infected with the virus, VA13 fibroblasts, which are transformed by the SV40 TAg, were used as positive controls. HCT116, which has mutated beta-catenin, and SW480, which is a model of CIN, were also used as controls. The genomes of the Mad-1 and Delta98 strains were transfected into cells. As negative controls we used pUC or no plasmids. Cells were collected at 0, 7, 14, and 21 days after transfection. PCR was used for the detection of TAg and the regulatory region DNA sequences at different time frames and Southern blot of whole genomic extracts for viral DNA integration into the host genome. Immunofluorescence and Western blot were performed for TAg, viral capsid proteins, and nuclear beta-catenin expressions, whereas coimmunoprecipitation was used to detect protein interactions. Karyotype analysis and electron microscopy were performed to seek chromosomal instability and cell abnormalities, respectively. Retention of viral sequences was observed for Mad-1- and Delta98-transfected RKO cells at all time frames with PCR only, whereas Southern blot analysis showed nonintegrated sequences at T7 alone. TAg and capsid protein expressions, as well as increased p53 and nuclear beta-catenin, were observed between T0 and T7 for Mad-1 and Delta98 alone. Also, interaction between TAg and both p53 and beta-catenin was also observed between T0 and T7. Chromosomal instability, characterized by chromosomal breakage, dicentric chromosomes, and increasing ploidy, was observed at all time frames for Mad-1 and Delta98, as well as cell abnormalities. In conclusion, we demonstrate that JC virus Mad-1 and Delta98 are able to induce chromosomal instability in colonic cells with a hit and run mechanism that involves an early interaction with beta-catenin and p53.