Influence of polymorphisms in ERCC5, XPA and MTR DNA repair and synthesis genes in B-cell lymphoma risk. A case-control study in Spanish population.

PURPOSE: Functions pertaining to DNA repair and synthesis are believed to play a critical role in cancer development and seem to be affected by genetic polymorphisms. Herein we performed a case-control study evaluating the influence of three single nucleotide polymorphisms (SNPs) in XPA, ERCC5 and MTR [rs1800975 (G-4A), rs17655 (Asp1104His) and rs1805087 (A2756G), respectively] in lymphoma risk. METHODS: Genotype distributions were studied in 213 lymphoma Caucasian patients (193 non-Hodgkin/NHL and 20 Hodgkin lymphoma/HL) and 214 controls, residents in a region of Southeast Spain. RESULTS: No significant differences were observed in the genotype distributions between cases and controls for the studied SNPs. This lack of association was also observed when stratifying for gender or lymphoma type. CONCLUSION: Our results suggest that the rs1800975, rs17655 and rs1805087 SNPs in DNA repair and synthesis of genes do not seem to play a major role in lymphoma susceptibility.

Colorectal serrated adenocarcinoma shows a different profile of oncogene mutations, MSI status and DNA repair protein expression compared to conventional and sporadic MSI-H carcinomas.

Molecular characterization has been extensively studied in serrated polyps but very little is known in serrated adenocarcinomas (SACs). We analyzed the incidence of KRAS, BRAF and PIK3CA mutations, microsatellite instability (MSI) status and loss of the DNA repair proteins MLH1, MSH2, MSH6 and MGMT in a series of 89 SAC, 81 matched conventional carcinomas (CC) and 13 sporadic colorectal cancer showing histological and molecular features of high-level MSI (sMSI-H). Our results demonstrate that KRAS are more prevalent than BRAF mutations in SAC (42.7% vs. 25.8%; p = 0.011) being the KRAS-mutated cases even more abundant in SAC displaying adjacent serrated adenomas (51%). G12D and E545K are the most common KRAS and PIK3CA mutations found in SAC, respectively. SAC show higher frequency of MGMT loss compared to CC (50.6% vs. 25.3%; p = 0.001) especially in distal colon/rectum (60.0% vs. 21.6%; p = 0.0009). SAC differ from sMSI-H in terms of KRAS and BRAF mutation prevalence, MSI status and MLH1 expression (p = 0.0003, p < 0.0001, p < 0.0001, p < 0.001, respectively). SACs are more often KRAS-mutated and microsatellite stable and display different molecular and immunohistochemical characteristics compared to CC and sMSI-H.

Tinción inmunohistoquímica p16 en carcinomas epidermoides del área genital y extragenital / Immunohistochemical Staining of p16 in Squamous Cell Carcinomas of the Genital and Extragenital Area

Introducción: La proteína p16 es una proteína supresora tumoral. El objetivo del estudio era comprobar si la tinción p16 se relaciona con la presencia de papilomavirus (subtipos mucosos o α, VPH-mc) en carcinomas epidermoides (CE) extragenitales (como ocurre en el cérvix y en CE genitales). Material y método: Se realizó tinción inmunohistoquímica con p16 a diversas lesiones incluidas en parafina del área genital (8 condilomas, tres CE intraepidérmicos y 7 CE invasores) y del área extragenital (20 CE intraepidérmicos tipo enfermedad de Bowen [EB] y 10 CE invasores). La detección de VPH-mc se realizó mediante reacción en cadena de la polimerasa (PCR). Resultados: En el área genital la tinción p16 fue negativa en los condilomas y positiva en los tres CE intraepidérmicos y en dos CE invasores (29%). Se detectó VPH-mc en 6 condilomas y dos CE intraepidérmicos (100%, excluyendo tres lesiones que no se pudieron estudiar con PCR) y en los dos CE invasores positivos para p16. En el área extragenital la tinción p16 fue positiva en 19 EB (95%) y en dos CE invasores (20%). Se detectó VPH-mc en 4 EB (tinción p16 positiva) y en un CE invasor (p16 negativa). En los CE intraepidérmicos la tinción p16 fue útil para objetivar si existían focos de microinfiltración dérmica o invasión de estructuras anexiales normales. Conclusiones: Según nuestros resultados la positividad de p16 es independiente de la detección de VPH en los CE extragenitales, al contrario de lo observado en CE genitales. En el área extragenital la pérdida de proteína p16 en los CE invasores respecto a los CE intraepidérmicos indicaría progresión tumoral (AU)

Background and objectives: Positive immunostaining for the tumor suppressor protein p16 is associated with the presence of mucosal or alfa subtypes of human papillomavirus (HPV) in cervical and genital squamous cell carcinoma (SCC). The aim of this study was to determine whether p16 immunostaining is also associated with mucosal HPV in extragenital SCC. Material and methods: Paraffin sections of lesions located in the genital region (8 genital warts, 3 intraepidermal SCCs, and 7 invasive SCCs) and extragenital area (29 intraepidermal SCCs corresponding to Bowen disease and 10 invasive SCCs) were stained for p16 by immunohistochemistry. Mucosal HPV was detected by polymerase chain reaction (PCR). Results: In the genital area, p16 immunostaining was negative in genital warts and positive in all 3 intraepidermal SCCs and 2 invasive SCCs (29%). Mucosal HPV was detected in 6 genital warts and 2 intraepidermal SCCs (100% after exclusion of 3 lesions that could not be analyzed by PCR) and in the 2 invasive SCCs that were positive for p16. In the extragenital area, 19 intraepidermal SCCs (95%) and 2 invasive SCCs (20%) were immunopositive for p16. Mucosal HPV was detected in 4 intraepidermal SCCs (p16 immunopositive) and 1 invasive SCC (p16 immunonegative). In intraepidermal SCCs, p16 immunostaining facilitated the identification of dermal microinfiltration or invasion of normal skin appendages. Conclusions: According to our results, unlike in genital SCCs, p16 immunopositivity is independent of the presence of HPV in extragenital SCCs. Compared with intraepidermal SCCs, the absence of p16 protein in invasive SCCs in the extragenital area would indicate progression of the disease (AU)

[Immunohistochemical staining of p16 in squamous cell carcinomas of the genital and extragenital area]. / Tinción inmunohistoquímica p16 en carcinomas epidermoides del área genital y extragenital.

BACKGROUND AND OBJECTIVES: Positive immunostaining for the tumor suppressor protein p16 is associated with the presence of mucosal or αsubtypes of human papillomavirus (HPV) in cervical and genital squamous cell carcinoma (SCC). The aim of this study was to determine whether p16 immunostaining is also associated with mucosal HPV in extragenital SCC. MATERIAL AND METHODS: Paraffin sections of lesions located in the genital region (8 genital warts, 3 intraepidermal SCCs, and 7 invasive SCCs) and extragenital area (29 intraepidermal SCCs corresponding to Bowen disease and 10 invasive SCCs) were stained for p16 by immunohistochemistry. Mucosal HPV was detected by polymerase chain reaction (PCR). RESULTS: In the genital area, p16 immunostaining was negative in genital warts and positive in all 3 intraepidermal SCCs and 2 invasive SCCs (29%). Mucosal HPV was detected in 6 genital warts and 2 intraepidermal SCCs (100% after exclusion of 3 lesions that could not be analyzed by PCR) and in the 2 invasive SCCs that were positive for p16. In the extragenital area, 19 intraepidermal SCCs (95%) and 2 invasive SCCs (20%) were immunopositive for p16. Mucosal HPV was detected in 4 intraepidermal SCCs (p16 immunopositive) and 1 invasive SCC (p16 immunonegative). In intraepidermal SCCs, p16 immunostaining facilitated the identification of dermal microinfiltration or invasion of normal skin appendages. CONCLUSIONS: According to our results, unlike in genital SCCs, p16 immunopositivity is independent of the presence of HPV in extragenital SCCs. Compared with intraepidermal SCCs, the absence of p16 protein in invasive SCCs in the extragenital area would indicate progression of the disease.

Papel del citocromo P450 en la farmacocinética y en la farmacogenética de los fármacos antihipertensivos / Role of CYP450 in pharmacokinetics and pharmacogenetics of antihypertensive drugs

Introducción Los fármacos antihipertensivos son metabolizados principalmente por enzimas de la familia del citocromo P450 (CYP450). La respuesta al tratamiento antihipertensivo cuya base genética está empezando a conocerse está sujeta a diferencias interindividuales en los pacientes. Objetivo El objetivo de esta revisión es documentar la metabolización de los fármacos antihipertensivos por enzimas del citocromo P450 e identificar cuáles pueden ser los polimorfismos más relevantes en los genes que codifican para estas enzimas con el fin de facilitar futuros estudios sobre la farmacogenética de la hipertensión. Métodos Se realizó una búsqueda por palabras clave en las bases de datos bibliográficas Pubmed, Rxlist y Medscape. Así mismo se consultaron las bases públicas de polimorfismos genéticos PharmGKB, NCBI y la página del comité para la nomenclatura de alelos del CYP450.ResultadosLas enzimas CYP2D6, CYP2C9, CYP2D19 y CYP3A4 participan en el metabolismo de la mayoría de los fármacos antihipertensivos. Teniendo en cuenta la frecuencia alélica en la población y la variabilidad en la respuesta clínica asociada sería interesante el estudio de los siguientes alelos: CYP2D6 *2, *4, deleción y duplicación; CYP2C9*2 y *3, CYP2C19 *2 y CYP3A4*1B.ConclusionesEl estudio de polimorfismos en los genes del citocromo P450 puede contribuir a una terapia individualizada en el tratamiento antihipertensivo (AU)

Introduction Antihypertensive drugs are principally metabolised by enzymes of the P450 (CYP450) cytochrome family. Response to hypertensive treatment whose genetic basis is beginning to be known presents inter-individual differences among patients. Objective The aim of this review is to document the role of cytochrome enzymes P450 in the metabolising process of antihypertensive drugs, and to identify the most relevant polymorphisms in genes that code for these enzymes in order to facilitate future studies in hypertension pharmacogenetics. Methods: A keyword search was performed in the following literature databases: Pubmed, Rxlist and Medscape. Genetic polymorphism public databases were also consulted (PharmGKB, NCBI and the CYP450 allele nomenclature committee web page. Results Enzymes CYP2D6, CYP2C9, CYP2D19 and CYP3A4 participate in the metabolising process of most antihypertensive drugs. Considering the allelic frequency in the population and the variability in the clinical response associated with genetic polymorphism, we find the study of the following alleles CYP2D6 *2, *4, deletion and duplication; CYP2C9*2 and *3, CYP2C19 *2 and CYP3A4*1B to be of crucial importance. Conclusions The study of polymorphisms in P450 cytochrome genes may contribute to an individualised therapy in the treatment against hypertension (AU)

Role of CYP450 in pharmacokinetics and pharmacogenetics of antihypertensive drugs.

INTRODUCTION: Antihypertensive drugs are principally metabolised by enzymes of the P450 (CYP450) cytochrome family. Response to hypertensive treatment whose genetic basis is beginning to be known presents inter-individual differences among patients. OBJECTIVE: The aim of this review is to document the role of cytochrome enzymes P450 in the metabolising process of antihypertensive drugs, and to identify the most relevant polymorphisms in genes that code for these enzymes in order to facilitate future studies in hypertension pharmacogenetics. METHODS: A keyword search was performed in the following literature databases: Pubmed, Rxlist and Medscape. Genetic polymorphism public databases were also consulted (PharmGKB, NCBI and the CYP450 allele nomenclature committee web page. RESULTS: Enzymes CYP2D6, CYP2C9, CYP2D19 and CYP3A4 participate in the metabolising process of most antihypertensive drugs. Considering the allelic frequency in the population and the variability in the clinical response associated with genetic polymorphism, we find the study of the following alleles CYP2D6 *2, *4, deletion and duplication; CYP2C9*2 and *3, CYP2C19 *2 and CYP3A4*1B to be of crucial importance. CONCLUSIONS: The study of polymorphisms in P450 cytochrome genes may contribute to an individualised therapy in the treatment against hypertension.

Association of polymorphism in FcGR3A gene and progression of low-grade precursor lesions of cervical carcinoma.

Polymorphisms in receptors of the constant part of antibodies (FcR) have been associated with susceptibility to disease and viral infections but have not been studied in cervical carcinogenesis. The distribution of the polymorphism V158F (rs396991) in FcGR3A in cervical smears was detected in a group of 84 women with stable or regressed low-grade squamous intraepithelial lesions (group I) and a group of 54 women with high-grade squamous intraepithelial lesions (HSIL) (group II). Human papillomavirus (HPV) genotyping was also performed. In 27.4% of women from group I, FF genotype was found, whereas this genotype was observed in 51.9% of patients in group II (p = 0.003; odds ratio = 2.856 (95% confidence interval = 1.4-5.8)). When only women infected with high-risk HPV were analyzed these differences were found to be even higher (p = 0.0013; odds ratio = 3.8 (95% confidence interval = 1.7-8.8)). FF genotype in FcGR3A gene seems to be associated with increased risk of low-grade squamous intraepithelial lesions to HSIL progression suggesting that its presence may play a role in HPV tolerance, persistent infection, and HSIL development.

Immunohistochemical evaluation of ProEx C in human papillomavirus-induced lesions of the cervix.

AIMS: Considering the sparse information about the clinical utility of the novel immunohistochemical marker ProEx C in histological sections, a decision was taken to study the pattern of ProEx C expression in normal/benign cervical epithelium (N/B), low-grade squamous intraepithelial lesion (LGSIL) and high-grade squamous intraepithelial lesion (HGSIL), as well as the association of ProEx C expression with human papillomavirus (HPV) genotypes. METHODS: 100 cervical samples, including 21 N/B cervices, 16 LGSILs, 61 HGSILs and two cervical invasive carcinomas, were obtained from conisation and hysterectomy. Surgical specimens were arranged in three tissue microarrays and stained for ProEx C. Ninety-three samples were HPV genotyped. Genotyping was performed by DNA amplification and hybridisation with genotype-specific probes on a low-density DNA array. RESULTS: ProEx C-positive expression in more than the lower third of the epithelium was observed in 14.3% of N/B, 62.5% of LGSIL and 90.2% of HGSIL. Seventy percent of HPV positivity was found in cases with expression in more than the lower third of the epithelium. Of 31 cases that were positive for HPV16, 16.1% showed ProEx C expression restricted to one or two basal layers, and 83.9% showed ProEx C expression in more than the lower third of the epithelium. CONCLUSIONS: ProEx C is significantly associated with HPV16 infection and is a useful adjunct in the identification of LGSIL and HGSIL in histological sections when expressed in more than the lower third of the epithelium.

Correlation between the effect of the anti-neoplastic ether lipid 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine on the membrane and the activity of protein kinase Calpha.

The antineoplastic ether phospholipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phophocholine (ET-18-OCH3) was incorporated into dimyristoylglycerophosphocholine (Myr2Gro-PCho)/dimyristoylglycerophosphoserine (Myr2Gro-PSer) (4 : 1 molar ratio) mixtures. Electron microscopy showed that the addition of ET-18-OCH3 reduced the size of the vesicles. Small vesicles could be detected even at 60 mol% ET-18-OCH3. Sedimentation studies showed the increasing presence of phospholipids in the supernatant, while turbidity measurements indicated a decrease in absorbance as the ET-18-OCH3 concentration was increased. These findings may be explained by the formation of small vesicles and/or mixed micelles. Infrared spectroscopy showed that at 60 mol% the fluidity of the membrane was considerably increased at temperatures below the phase transition, with only a small increase in the proportion of gauche isomers after the gel-to-fluid phase transition of this sample. On the other hand, protein kinase Calpha (PKCalpha) activity progressively decreased when ET-18-OCH3 was incorporated into multilamellar vesicles, reaching a minimum value at 20 mol%, this inhibition being attributed to the modification of the membrane produced by a cone-shaped molecule. At higher concentrations, however, ET-18-OCH3 activated the enzyme with a maximum being attained at 50 mol%. This activation being attributed to the formation of small vesicles and/or micelles. At still higher concentrations of ET-18-OCH3 the enzyme was once again inhibited, inhibition being almost complete at 80 mol%. When PKC was assayed using large unilamellar vesicles a slight activation was observed at very low ET-18-OCH3 concentrations.

Identification of the phosphatidylserine binding site in the C2 domain that is important for PKC alpha activation and in vivo cell localization.

The C2 domain of classical PKCs binds to membranes through Ca(2+) bridging to phosphatidylserine as recently observed through X-ray diffraction of the isolated domain. Additionally, it has been proposed that N189, T251, R216, and R249A interact directly with phosphatidylserine [Verdaguer, N., et al. (1999) EMBO J. 18, 6329-6338]. When these four residues were mutated to Ala to determine their role in PKC binding to phospholipid membranes, PKC activation, and in its in vivo localization, the results revealed that they were very important for the activation of full-length PKCalpha. N189, in particular, was involved in the activation of the enzyme after its interaction with PS, since its mutation to Ala did not decrease the level of membrane binding but did prevent full enzyme activation. On the other hand, mutations R216A, R249A, and T251A affected both membrane binding and enzyme activation, although T251A had the most drastic effect, suggesting that the protein interactions with the carbonyl groups of the phospholipid are also a key event in the activation process. Taken together, these results show that the four residues located near the calcium binding site are critical in phosphatidylserine-dependent PKCalpha activation, in which N189 plays an important role, triggering the enzyme activation probably by interacting with neighboring residues of the protein when lipid binding occurs. Furthermore, these results provide strong evidence for better defining one of the two phosphatidylserine isomer models proposed in the previous crystallographic report.

The C2 domain of protein kinase calpha is directly involved in the diacylglycerol-dependent binding of the C1 domain to the membrane.

Protein kinase Calpha (PKCalpha), which is known to be critical for the control of many cellular processes, was submitted to site-directed mutagenesis in order to test the functionality of several amino acidic residues. Thus, D187, D246 and D248, all of which are located at the Ca(2+) binding site of the C2 domain, were substituted by N. Subcellular fractionation experiments demonstrated that these mutations are important for both Ca(2+)-dependent and diacylglycerol-dependent membrane binding. The mutants are not able to phosphorylate typical PKC substrates, such as histone and myelin basic protein. Furthermore, using increasing concentrations of dioleylglycerol, one of the mutants (D246/248N) was able to recover total activity although the amounts of dioleylglycerol it required were larger than those required by wild type protein. On the other hand, the other mutants (D187N and D187/246/248) only recovered 50% of their activity. These data suggest that there is a relationship between the C1 domain, where dioleylglycerol binds, and the C2 domain, and that this relationship is very important for enzyme activation. These findings led us to propose a mechanism for PKCalpha activation, where C1 and C2 domains cannot be considered independent membrane binding modules.