p53 expression in colorectal carcinoma in relation to histopathological features in Ugandan patients

Background

It has been shown that colorectal carcinoma is increasing in incidence in African countries. This could be due to change in life style. Molecular pathogenesis of colorectal cancer commonly involves mutation in p53 gene which leads to expression of p53 protein in tumor cells. Expression of p53 protein has been associated with poor clinical outcome and reduced survival in patients.

Objective

This was a retrospective laboratory based study carried out in the Department of Pathology Makerere University, Kampala, Uganda. The aim of the study was to evaluate the expression of p53 protein in colorectal carcinoma in Ugandan patients, specifically its association with histological types, degree of differentiation, sites of the tumor and demographic characteristics of the patients.

Methods

Immunohistochemistry was carried out on 109 patient''s paraffin embedded tissue blocks of colorectal carcinoma diagnosed in the Pathology Department, Faculty of Medicine Makerere University Kampala during the period 1995 to 2005. The indirect immunoperoxidase method using monoclonal antibody p53 DO-7 and Envision ⁺ Dual link system-HRP to detect p53 expression was used. Haematoxylin and eosin stain was used for evaluation of histological types and degree of differentiation of the tumors. Topography of the tumors and demographic data were obtained from accompanying histological request forms.

Results

Out of 109 patient''s tissue blocks that were studied, 61 cases (56%) expressed p53 protein in the nucleus of malignant cells. Right sided colonic tumors were commoner (53.2%) than left sided colonic tumors (46.8%). p53 protein was expressed more in left sided colonic tumors with a significant difference (p<0.05), it was also expressed more in well differentiated tumors and non mucinous adenocarcinomas but with no significant difference (p>0.05). p53 expression was not affected by age or sex.

Conclusion

Frequency of p53 protein expression in Ugandan patients did not differ from that reported in the other parts of the world. It was expressed more in the left sided colonic tumors and this could support the hypothesis that right and left colonic tumors could have different pathogenesis and probably also responsible for difference in prognosis in these two topographic sites.     

Allele-specific regulation of DISC1 expression by miR-135b-5p

Disrupted-in-schizophrenia-1 (DISC1) gene has been established as a risk factor for various neuropsychiatric phenotypes. Both coding and regulatory variants in DISC1 have been identified and associated with these phenotypes in genetic studies. MicroRNAs (miRNAs) are important regulators of protein coding genes. Since the miRNA-mRNA target recognition mechanism is vulnerable to disruption by DNA polymorphisms, we investigated whether polymorphisms in the DISC1 3′UTR affect binding of miRNAs and lead to allele-specific regulation of DISC1. We identified four predicted polymorphic miRNA target sites in the DISC1 3′UTR, and demonstrated that miR-135b-5p regulates the level of DISC1 mRNA. Moreover, DISC1 regulation by miR-135b-5p is allele specific: miR-135b-5p only binds to the major allele (A) of rs11122396, not to the minor allele (G). Thus, the G allele may be functionally related to the DISC1-associated phenotypes by abolishing regulation by miR-135b-5p, leading to elevated DISC1 levels.     

microRNA-155 acts as an oncogene by targeting the tumor protein 53-induced nuclear protein 1 in esophageal squamous cell carcinoma

MicroRNA-155 (miR-155) is overexpressed in many human cancers; however, the function of miR-155 is largely unknown in esophageal squamous cell carcinoma (ESCC). In the present study, we found that miR-155 is dramatically increased in ESCC tissues compared with the paired adjacent normal tissues, which suggested that miR-155 acts as an oncogene in ESCC. We predicted that tumor protein p53-induced nuclear protein 1 (TP53INP1) is a candidate target gene of miR-155 given that miR-155 expression decreased mRNA and protein levels of TP53INP1 as determined by RT-PCR and Western blot analysis. In addition, miR-155 and TP53INP1 showed a negative relation in ESCC tissues. Dual luciferase-based reporter assay indicated direct regulation of TP53INP1 by miR-155. Furthermore, we demonstrated that RNA interference of TP53INP1 increased the proliferation and colonies formation of EC-1 cells. Up-regulation of TP53INP1 abrogated miR-155 induced growth in EC-1 cells and mutation of TP53INP1 in 3’-UTR restored the effects when co-transfected with miR-155. We also indicated that overexpression of miR-155 significantly promoted the proliferation of EC-1 cells in vitro and the development of tumors in nude mice. Taken together, our study reveals that miR-155 acts as an oncogene by targeting TP53INP1 in ESCC.     

Genetic polymorphism of p53, but not GSTP1, is association with susceptibility to esophageal cancer risk - A Meta-Analysis

A number of studies have evaluated two functional polymorphisms on p53 Arg72Pro and GSTP1 Ile105Val, in relation to esophageal cancer susceptibility. However, the results remain conflicting rather than conclusive. This meta-analysis on 2919 cases and 4074 controls for p53 Arg72Pro and 1885 cases and 2194 controls for GSTP1 Ile105Val from 13 published case-control studies showed that no significant general main effects for GSTP1 Ile105Val on esophageal cancer risk. However, we found that the p53 Arg72Pro was associated with an increased risk of esophageal cancer ((Pro/Arg +Pro/Pro) versus Arg/Arg: OR=1.20, 95%CI=1.06-1.36) without any between-study heterogeneity.In the stratified analysis by ethnicity, we found that the increased esophageal cancer risk associated with p53 Arg72Pro polymorphism was more evident in Asian group ((Pro/Arg +Pro/Pro) versus Arg/Arg: OR=1.35, 95%CI=1.14-1.60, P=0.09 for heterogeneity test), although we still failed to find any significant association between GSTP1 Ile105Val polymorphism and esophageal cancer risk in different ethnicity. These results suggest that p53 Arg72Pro polymorphism, but not GSTP1 Ile105Val, may contribute to esophageal cancer development, especially in Asian. Additional well-designed large studies were required for the validation of this association.     

Qiliqiangxin improves cardiac function and attenuates cardiac remodeling in rats with experimental myocardial infarction

Objective: It has been reported that Qiliqiangxin (QL), a traditional Chinese medicine compound, could inhibit cardiac hypertrophy and remodeling, and improve cardiac function. However, whether and how it reverses cardiac remodeling in rats post myocardial infarction (MI) remains unknown. This study aims to explore related mechanisms linked with cardiac function improvement and attenuation of cardiac remodeling by QL in rats with experimental MI. Methods: MI was induced by ligation of left anterior descending coronary artery (LAD) in male Sprague-Dawley rats. Rats with LVEF < 50% at four weeks after procedure were treated for another 6 weeks with placebo, QL and captopril. Echocardiography and plasma NT-proBNP were measured at the end of study, and histological studies were performed. Protein expressions of Neuregulin-1 (NRG-1), total-Akt, phospho-Akt (Ser473), hydroxy-HIF-1α (Pro564), VEGF, Bax, Bcl-2 and Caspase 3 were examined by Western blot. mRNA expression of NRG-1 and p53 was detected by real-time PCR. Results: Compared with the placebo group, QL improved cardiac function, reduced left ventricular dimension, inhibited interstitial inflammation and fibrosis, increased neovascularization, and attenuated cardiomyocyte apoptosis. Meanwhile QL significantly upregulated the expression of HIF-1α, VEGF, enhanced phosphorylation of Akt, decreased the ratio of Bax/Bcl-2 and Caspase 3 expression. Furthermore, we observed upregulation of NRG-1 and downregulation of p53 after QL treatment. Conclusion: Our data suggest that the beneficial effects of QL on improving cardiac function and attenuating cardiac remodeling post MI are associated with angiogenesis enhancement and apoptosis inhibition, which may be mediated via activation of NRG-1/Akt signaling and suppression of p53 pathway.     

The effect of adenovirus-conjugated NDRG2 on p53-mediated apoptosis of hepatocarcinoma cells through attenuation of nucleotide excision repair capacity

NDRG2 mRNA and protein levels can be upregulated in a p53-dependent manner. NDRG2 enhances p53-mediated apoptosis, whereas overexpression of NDRG2 suppresses tumor cell growth, regardless of whether p53 is mutated. However, the complicated mechanism by which NDRG2 suppresses tumor cell growth and enhances apoptosis mediated by p53 is not fully understood. Here, we demonstrated that Ad-NDRG2 enhanced the apoptosis of HepG2 cells (wild-type p53). Additionally, Ad-NDRG2 combined with rAd-p53 enhanced the apoptosis of Huh7 cells (mutant p53) after chemotherapy, and the expression of the ERCC6 gene (Cockayne syndrome group B protein gene) was suppressed in this process. Ad-NDRG2 combined with rAd-p53 induced the apoptosis of tumor cells (HepG2 and Huh7 cells); however, apoptosis was attenuated after transfection with ERCC6. Our results indicate that Ad-NDRG2 enhances the p53-mediated apoptosis of hepatocarcinoma cells (HepG2 and Huh7) by attenuating the nucleotide excision repair capacity (i.e., by downregulating ERCC6), and ERCC6 is a NDRG2-inducible target gene that is involved in the p53-mediated apoptosis pathway.     

An unusual clinical severity of 16p11.2 deletion syndrome caused by unmasked recessive mutation of CLN3

With the introduction of array comparative genomic hybridization (aCGH) techniques in the diagnostic setting of patients with developmental delay and congenital malformations, many new microdeletion syndromes have been recognized. One of these recently recognized microdeletion syndromes is the 16p11.2 deletion syndrome, associated with variable clinical outcomes including developmental delay, autism spectrum disorder, epilepsy, and obesity, but also apparently normal phenotype. We report on a 16-year-old patient with developmental delay, exhibiting retinis pigmentosa with progressive visual failure from the age of 9 years, ataxia, and peripheral neuropathy. Chromosomal microarray analysis identified a 1.7-Mb 16p11.2 deletion encompassing the 593-kb common deletion (∼29.5 to ∼30.1 Mb; Hg18) and the 220-kb distal deletion (∼28.74 to ∼28.95 Mb; Hg18) that partially included the CLN3 gene. As the patient''s clinical findings were different from usual 16p11.2 microdeletion phenotypes and showed some features reminiscent of juvenile neuronal ceroid-lipofuscinosis (JNCL, Batten disease, OMIM 204200), we suspected and confirmed a mutation of the remaining CLN3 allele. This case further illustrates that unmasking of hemizygous recessive mutations by chromosomal deletion represents one explanation for the phenotypic variability observed in chromosomal deletion disorders.     

Apoptosis of leukocytes triggered by acute DNA damage promotes lymphoma formation

Apoptosis triggered by p53 upon DNA damage secures removal of cells with compromised genomes, and is thought to prevent tumorigenesis. In contrast, we provide evidence that p53-induced apoptosis can actively drive tumor formation. Mice defective in p53-induced apoptosis due to loss of its proapoptotic target gene, puma, resist γ-irradiation (IR)-induced lymphomagenesis. In wild-type animals, repeated irradiation injury-induced expansion of hematopoietic stem/progenitor cells (HSCs) leads to lymphoma formation. Puma^−/− HSCs, protected from IR-induced cell death, show reduced compensatory proliferation and replication stress-associated DNA damage, and fail to form thymic lymphomas, demonstrating that the maintenance of stem/progenitor cell homeostasis is critical to prevent IR-induced tumorigenesis.     

Novel progranulin mutations with reduced serum-progranulin levels in frontotemporal lobar degeneration

Frontotemporal lobar degeneration (FTLD) is a progressive neurodegenerative disease with an age at onset generally below 65 years. Mutations in progranulin (GRN) have been reported to be able to cause FTLD through haploinsufficiency. We have sequenced GRN in 121 patients with FTLD and detected six different mutations in eight patients: p.Gly35Glufs*19, p.Asn118Phefs*4, p.Val200Glyfs*18, p.Tyr294*, p.Cys404* and p.Cys416Leufs*30. Serum was available for five of the mutations, where the serum-GRN levels were found to be >50% reduced compared with FTLD patients without GRN mutations. Moreover, the p.Cys416Leufs*30 mutation segregated in an affected family with different dementia diagnoses. The mutation frequency of GRN mutation was 6.6% in our FTLD cohort.     

Germline copy number variation of genes involved in chromatin remodelling in families suggestive of Li-Fraumeni syndrome with brain tumours

Germline alterations of the tumour suppressor TP53 gene are detected approximately in 25% of the families suggestive of Li-Fraumeni syndrome (LFS), characterised by a genetic predisposition to a wide tumour spectrum, including soft-tissue sarcomas, osteosarcomas, premenopausal breast cancers, brain tumours, adrenocortical tumours, plexus choroid tumours, leukaemia and lung cancer. The aim of this study was to determine the contribution of germline copy number variations (CNVs) to LFS in families without detectable TP53 mutation. Using a custom-designed high-resolution array CGH, we evaluated the presence of rare germline CNVs in 64 patients fulfilling the Chompret criteria for LFS, but without any detectable TP53 alteration. In 15 unrelated patients, we detected 20 new CNVs absent in 600 controls. Remarkably, in four patients who had developed each brain tumour, the detected CNV overlap the KDM1A, MTA3, TRRAP or SIRT3 genes encoding p53 partners involved in histone methylation or acetylation. Focused analysis of SIRT3 showed that the CNV encompassing SIRT3 leads to SIRT3 overexpression, and that in vitro SIRT3 overexpression prevents apoptosis, increases G2/M and results in a hypermethylation of numerous genes. This study supports the causal role of germline alterations of genes involved in chromatin remodelling in genetic predisposition to cancer and, in particular, to brain tumours.     

p53 Codon 72 Genetic Polymorphism in Asthmatic Children: Evidence of Interaction With Acid Phosphatase Locus 1

Several lines of evidence are implicating an increased persistence of apoptotic cells in patients with asthma. This is largely due to a combination of inhibition, or defects in the apoptotic process and/or impaired apoptotic cell removal mechanisms. Among apoptosis-inducing genes, an important role is played by p53. In the present study, we have investigated the possible relationship between p53 codon 72 polymorphism and asthma and the interaction with ACP1, a genetic polymorphism involved in the susceptibility to allergic asthma. We studied 125 asthmatic children and 123 healthy subjects from the Caucasian population of Central Italy. p53 codon 72 and ACP1 polymorphisms were evaluated using a restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) method. There is a statistically significant association between p53 codon 72 polymorphism and allergic asthma: Arg/Arg genotype is more represented in asthmatic patients than in controls (P=0.018). This association, however, is present in subjects with low ACP1 activity A/A and A/B only (P=0.023). The proportion of children with A/A and A/B genotype carrying Arg/Arg genotype is significantly high in asthmatic children than in controls (OR=1.941; 95% C.I. 1.042-3.628). Our finding could have important clinical implications since the subjects with A/A and A/B genotypes of ACP1 carrying Arg/Arg genotype are more susceptible to allergic asthma than Pro/Pro genotype.     

Impact of p73 gene polymorphism on cancer susceptibility: a meta analysis

Previous studies examining the association between p73 G4A and gastric cancer risk have produced inconsistent results. The objective of this study was to clarify whether p73 G4A plays a major role in the development of gastric cancer. Studies that had examined the association between p73 G4A and gastric cancer risk were identified through PubMed, Science Direct, and CNKI. We selected eligible studies based on inclusion criteria. Odds ratios were estimated using distinct genetic models, and the heterogeneity between studies was explored using Cochran’s Q statistic along with the I² statistic. Overall, we found no evidence of a significant association between p73 G4A and risk of gastric cancer. A same trend was also indicated in subgroup analysis by ethnicity. The heterogeneity tests revealed that there was no significant heterogeneity across studies. Our meta-analysis indicates that p73 G4A might not have a major effect on risk of gastric cancer. A much larger study is required to validate our findings.     

Acute Toxicity of Opuntia Ficus Indica and Pistacia Lentiscus Seed Oils in Mice

Opuntia ficus indica and Pistacia lentiscus L. seeds are used in traditional medicine. The objective of this study was to investigate the toxicity of the fixed oil of Opuntia ficus indica and Pistacia lentiscus L. seeds in mice through determination of LD₅₀ values, and also the physicochemical characteristics of the fixed oil of these oils. The acute toxicity of their fixed oil were also investigated in mice using the method of Kabba and Berhens. The fixed oil of Pistacia lentiscus and Opuntia ficus indica seeds were extracted and analyzed for its chemical and physical properties such as acid value, free fatty acid percentage (% FFA), iodine index, and saponification value as well as refractive index and density. LD₅₀ values obtained by single doses, orally and intraperitoneally administered in mice, were respectively 43 ± 0,8 ;[40.7– 45.4 ] ml/kg body wt. p.o. and 2.72 ± 0,1 ;[2.52–2.92] ml/kg body wt. i.p. for Opuntia ficus indica ; and 37 ± 1 ;[34.4 − 39.8 ] ml/kg body wt. p.o. and 2.52 ± 0,2 ;[2.22 − 2.81 ] ml/kg body wt. i.p. for Pistacia lentiscus respectively. The yields of seed oil were respectively calculated as 20.25% and 10.41%. The acid and free fatty acid values indicated that the oil has a low acidity     

Deletions involving genes WHSC1 and LETM1 may be necessary,but are not sufficient to cause Wolf–Hirschhorn Syndrome

Wolf–Hirschhorn syndrome (WHS) is a complex genetic disorder caused by the loss of genomic material from the short arm of chromosome 4. Genotype–phenotype correlation studies indicated that the loss of genes within 4p16.3 is necessary for expression of the core features of the phenotype. Within this region, haploinsufficiency of the genes WHSC1 and LETM1 is thought to be a major contributor to the pathogenesis of WHS. We present clinical findings for three patients with relatively small (<400 kb) de novo interstitial deletions that overlap WHSC1 and LETM1. 3D facial analysis was performed for two of these patients. Based on our findings, we propose that hemizygosity of WHSC1 and LETM1 is associated with a clinical phenotype characterized by growth deficiency, feeding difficulties, and motor and speech delays. The deletion of additional genes nearby WHSC1 and LETM1 does not result in a marked increase in the severity of clinical features, arguing against their haploinsufficiency. The absence of seizures and typical WHS craniofacial findings in our cohort suggest that deletion of distinct or additional 4p16.3 genes is necessary for expression of these features. Altogether, these results show that although loss-of-function for WHSC1 and/or LETM1 contributes to some of the features of WHS, deletion of additional genes is required for the full expression of the phenotype, providing further support that WHS is a contiguous gene deletion disorder.     

Genetic alterations in quadruple malignancies of a patient with multiple sclerosis: their role in malignancy development and response to therapy

Multiple cancers represent 2.42% of all human cancers and are mainly double or triple cancers. Many possible causes of multiple malignancies have been reported such as genetic alterations, exposure to anti-cancer chemotherapy, radiotherapy, immunosuppressive therapy and reduced immunologic response. We report a female patient with multiple sclerosis and quadruple cancers of different embryological origin. Patient was diagnosed with stage III (T3, N1a, MO) medullary thyroid carcinoma (MTC), multicentric micropapillary thyroid carcinoma, scapular and lumbar melanomas (Clark II, Breslow II), and lobular invasive breast carcinoma (T1a, NO, MO). All tumors present in our patient except micropapillary thyroid carcinomas were investigated for gene alterations known to have a key role in cancer promotion and progression. Tumor samples were screened for the p16 alterations (loss of heterozygosity and homozygous deletions), loss of heterozygosity of PTEN, p53 alterations (mutational status and loss of heterozygosity) and mutational status of RET, HRAS and KRAS. Each type of tumor investigated had specific pattern of analyzed genetic alterations. The most prominent genetic changes were mutual alterations in PTEN and p53 tumor suppressors present in breast cancer and two melanomas. These co-alterations could be crucial for promoting development of multiple malignancies. Moreover the insertion in 4^th codon of HRAS gene was common for all tumor types investigated. It represents frameshift mutation introducing stop codon at position 5 which prevents synthesis of a full-length protein. Since the inactivated RAS enhances sensitivity to tamoxifen and radiotherapy this genetic alteration could be considered as a good prognostic factor for this patient.     

Target RNA capture and cleavage by the Cmr type III-B CRISPR–Cas effector complex

The effector complex of the Cmr/type III-B CRISPR (clustered regularly interspaced short palindromic repeat)–Cas (CRISPR-associated) system cleaves RNAs recognized by the CRISPR RNA (crRNA) of the complex and includes six protein subunits of unknown functions. Using reconstituted Pyrococcus furiosus Cmr complexes, we found that each of the six Cmr proteins plays a critical role in either crRNA interaction or target RNA capture. Cmr2, Cmr3, Cmr4, and Cmr5 are all required for formation of a crRNA-containing complex detected by native gel electrophoresis, and the conserved 5′ repeat sequence tag and 5′-OH group of the crRNA are essential for the interaction. Interestingly, capture of the complementary target RNA additionally requires both Cmr1 and Cmr6. In detailed functional studies, we determined that P. furiosus Cmr complexes cleave target RNAs at 6-nucleotide (nt) intervals in the region of complementarity, beginning 5 nt downstream from the crRNA tag and continuing to within ∼14 nt of the 3′ end of the crRNA. Our findings indicate that Cmr3 recognizes the signature crRNA tag sequence (and depends on protein–protein interactions with Cmr2, Cmr4, and Cmr5), each Cmr4 subunit mediates a target RNA cleavage, and Cmr1 and Cmr6 mediate an essential interaction between the 3′ region of the crRNA and the target RNA.