Genetic polymorphism of alcohol dehydrogenase 3 in digestive tract alcohol damage.

BACKGROUND/AIMS: Genetic polymorphism of enzymes involved in alcohol metabolism plays a relevant role in etiopathogenesis of alcohol disease. The aim of the present study was to find in the Polish population the ADH3 genotypes, which are likely to be responsible for higher susceptibility to alcohol disease of the liver and chronic alcohol pancreatitis. METHODOLOGY: The ADH3 genotype and ADH3*1 and ADH3*2 alleles frequency were examined in 266 patients. Genotyping of the ADH3 was performed using polymerase chain reaction-restriction fragment length polymorphism methods on white cell DNA. RESULTS: The genotype ADH3*1/ADH3*1 was found to be significantly more frequent in alcohol abusers compared to non-drinkers. The examinations of the group of alcohol abusers showed that the genotype ADH3*2/ADH3*2 occurred statistically significantly less frequently in patients with chronic pancreatitis than those without alimentary lesions and patients with cirrhosis. Thus it is likely to be the protective factor of chronic pancreatitis. CONCLUSIONS: The alleles ADH3*1 and genotype ADH3*1/ADH3*1 were significantly more frequent in men than in women, while alleles ADH3*2 and genotype ADH3*2/ADH3*2 were more common in women. This may account for rarer alcohol addiction observed in Polish women.

Survivin antiapoptotic gene expression as a prognostic factor in non-small cell lung cancer: in situ hybridization study.

Survivin is an inhibitor of apoptosis that plays a significant role in cell cycle regulation and is important for survival prognosis in many neoplasms. Survivin expression was assessed by in situ hybridization (ISH) in 60 consecutive patients (54 males and 4 females) with NSCLC treated between 1993 and 1997. The examined patients had IIB and IIIA stage according to TNM system. In all cases the chemotherapy with cisplatin and etoposide (2 cycles) was administered prior the surgery; in patients responding to the therapy one more cycle was applied. Survivin gene overexpression was observed in 35 patients (58.3%). There was no correlation between survivin mRNA level and histological type of tumor, stage of cell differentiation, stage of disease according to TNM classification, performance status according to WHO and number of chemotherapy regimens administered (p > 0.05). However, the correlation between survivin gene expression and response to the chemotherapy was statistically significant (p = 0.04). Statistical analysis showed that median survival in patients with survivin gene overexpression was shorter (14.0 months) as compared to patients with no expression (60.0 months; p = 0.00002). In survival assessment by means of Kaplan-Meier test, 14.3% of five-year survival was achieved in the former group versus 60% in the latter (p = 0.00003). Univariate analysis (log-rank test) showed that significant independent prognostic factors in NSCLC included: stage of the disease according to TNM classification (p = 0.006), response to chemotherapy (p = 0.005) and pattern of survivin gene expression (p = 0.00003). Multivariate analysis utilizing Cox's model showed that for survival assessment the stage according to TNM, response to the chemotherapy and survivin expression estimated by means of ISH are of statistical significance (p=0.00001). The calculated predictive values showed that ISH technique was quite accurate in assessment of five-year survival. Our data show that survivin expression may be used as a prognostic factor and a target for therapy.

K-ras gene point mutations in human endometrial carcinomas: correlation with clinicopathological features and patients' outcome.

In order to evaluate the role of K-ras gene point mutations in the progression of endometrial carcinoma, we applied the polymerase chain reaction/restriction-fragment-length polymorphism technique to 57 tumours surgically removed from women of Polish origin. We assessed the relationship between K-ras gene activation and clinicopathological features as well as patients' outcome. Mutational activation in codon 12 of the K-ras gene was detected in 8 out of 57 (14%) endometrial carcinomas, while in codon 13 of the K-ras gene no point mutations were noted. A correlation between the histological type of the tumour and codon 12 K-ras gene mutation was noted (P < 0.05; Fisher exact test). K-ras gene mutation was not related to the patients' age, surgical stage, histological grade or to the depth of myometrial invasion. A trend towards a poorer prognosis was noted during the follow-up of patients whose tumours had shown K-ras codon 12 point mutations, but the difference was not significant (P = 0.06; log-rank test). Our data indicate that point mutations in codon 12 of the K-ras gene are a rare event in human endometrial carcinomas. The lack of correlation between K-ras point mutations and clinicopathological features (except histological type) supports the hypothesis of a random activation of the K-ras gene in human neoplastic endometrium.

Detection of human papillomavirus types 16 and 18 in human neoplastic endometrium: lack of correlation with established prognostic factors.

HPV (types 16 and 18) DNA sequences are present in the majority of precancerous and cancerous lesions of the human uterine cervix. However, data concerning the involvement of HPVs infection in the pathogenesis of endometrial cancer are controversial. In the current study we investigated the frequency of the HPV types 16 and 18, detected by PCR amplification using the type 16- and 18-specific primers within the E7 Open Reading Frame (ORF) sequence, in 54 human endometrial carcinomas obtained from women of Polish origin. Moreover, we assessed the possible association of the HPV with the clinicopathological features of the cancer, patients' outcome as well as with the K-ras codon 12 gene point mutations. HPV type 16 was present in eleven out of 54 (20%) endometrial tumors, while HPV type 18 was detected only in three out of 54 (4%) neoplasms analyzed. HPV infection was not related either to the patients' age (r=0.11; p=0.428, Spearman correlation test) or to the clinicopathological parameters and patients' prognosis. A higher incidence of HPV 16/18 was detected in well (G1) differentiated than in moderately (G2) and poorly (G3) differentiated endometrial adenocarcinomas, but the difference was not statistically significant. Moreover, none of HPV-positive endometrial carcinomas harbored K-ras codon 12 gene point mutations. Our results suggest that some of the endometrial carcinomas are associated with HPV infection but the presence of the human papillamovirus types 16/18 is not related to the clinicopathological or prognostical features of the neoplasm.

Detection of CFTR gene mutations in patients suffering from chronic bronchitis.

BACKGROUND: The purpose of the study was to examine cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in patients suffering from chronic bronchitis. METHODS: Thirty-two patients admitted to the Department of Pulmonology, Lublin School of Medicine, Lublin, Poland between 1995 and 1996 due to chronic bronchitis were included in the study. Patients were analyzed for the eight most common mutations of the CFTR gene (DeltaF508, G542X, N1303K, 1717-1(GoA)), W1282X, G551D, R553X, and DeltaI507 by the reverse-hybridization method. RESULTS: CFTR gene mutations were found in five of 32 (16%) patients, all within the DeltaF508 region of the CFTR gene. All positive samples were obtained from patients heterozygous for the DeltaF508 mutation. The presence of the DeltaF508 mutation was considered statistically significant when our study group was compared to the study of Poland's general population (p <0.05 Fisher's exact test). CONCLUSION: Our results suggest there is an increased presence of the DeltaF508 point mutation of the CFTR gene in Polish patients suffering from chronic bronchitis.

"Low-risk" and "high-risk" HPV-infection and K-ras gene point mutations in human cervical cancer: a study of 31 cases.

To analyze the coexistence of human papilloma virus (HPV) infection and K-ras gene activation in cervical neoplasia, we investigated 31 (seven pre-invasive and 24 invasive) cervical carcinomas for "low-risk" (types 6 and 11) and "high-risk" (types 16 and 18) HPVs and K-ras point mutations using PCR-based technology. "Low-risk" HPVs were not detected in the group investigated; however, 20 of 31 (64%) cases were HPV 16 positive, while HPV 18 was found in only three (9.7%) samples (HPV 6/11 v. HPV 16/18, p < 0.0001; HPV 16 v. HPV 18, p < 0.0001; Fisher's exact test). There was a K-ras codon 12 point mutation in two of 31 (6.4%) neoplasms, with none of the cases showing a K-ras codon 13 point mutation. Two moderately differentiated squamous carcinomas showed K-ras exon 2 gene alterations. Interestingly, none of the pre-invasive cervical carcinomas displayed K-ras gene point mutations. The mean patient age did not differ significantly in the number of HPV-positive and -negative cases. A coexistence of "high-risk" human papillomavirus DNA with K-ras gene alterations was observed in three of 31 (9.7%) neoplasms (one IIA and two IB moderately differentiated cervical carcinomas). Our results suggest that "high-risk" HPVs coexist with K-ras gene alterations in a subset of moderately differentiated carcinomas of the cervix uteri.

Effect of tyrphostins on programmed cell death in colon adenocarcinoma cell line LS-180.

Programmed cell death is an important process in the regulation of cellular proliferation, rest, differentiation and death. It is a genetically controlled process with characteristic biochemical and morphological features. Apoptosis directly regulates tumorigenesis and its induction could be a useful method of cancer therapy. Cancer cells could be influenced by some factors which induce apoptosis. We investigated the influence of tyrphostins, that specifically inhibits protein tyrosine kinases and stops the cell cycle in apoptosis of the colon adenocarcinoma cell line LS180. We used them at the concentration of 1-10 microM for 24 and 48 hours. We detected apoptosis using techniques that monitor either biochemical and morphological features of this process, such as staining with 7-amino-actinomycin D, staining with Grünwald-Giemsa, TUNEL reaction, in situ hybridization and with immunoperoxidase staining procedures. We examined the expression of genes and proteins connected with programmed cell death (p53, c-myc, p21, bcl-2). We estimated the results by cytophotometry and documented them by colour photography. We found that tyrphostin rapidly inhibits the cell cycle, particularly at the concentration of 5 microM. The expression of genes and proteins was strongly correlated with the increased apoptotic cell death conforming to the results of TUNEL and staining methods.

Expression of p21 and bcl-2 proteins and p53 mRNA in surgically resected preparations of non-small cell lung cancer (stage IIIA) after etoposide and cisplatin chemotherapy.

Apoptosis which is also a called programmed cell death plays an important role during development, homeostasis and in many diseases such as cancer. Apoptosis is a genetically encoded cell death program defined by characteristic morphological and biochemical features. It is well recognized as a distinct pathologic mechanism in tumours responding to anticancer therapies. Many genes play an important role in this process. We evaluated an expression of the tumour supressor gene p53 and proteins p21 and bcl-2 in non-small cell lung cancer. We examined resected tumour tissues from 30 patients who received neoadjuvant chemotherapy. As a control we assessed tissues from patients treated without chemotherapy. Histological slides of the resected tumours were evaluated by TUNEL, in situ hybridisation and with immunoperoxidase staining procedure. The results were documented by photography. We examined the level of extinction using cytophotometry. In conclusion, preoperative chemotherapy induces apoptosis in cancer cells. The level of p53 correlates with the acceleration of TUNEL reaction. The loss of bcl-2 expression correlated with an increased apoptotic cell death. There was an increased p21 protein expression in the examined cancer tissues after chemotherapy.

Detection of K-ras mutations in cancerous lesions of human endometrium.

The many genetic changes associated with human carcinogenesis include the activation and/or inactivation of various genes. Polymerase-chain reaction and single-strand conformation polymorphism analysis [PCR-SSCP] was used to detect alterations at exon 1 of the K-ras gene in 20 lesions of human endometrium. Six cases of endometrial hyperplasia, 13 of endometrial carcinoma and one of endometrial metastasis of ovarian cancer were analyzed. Mutations at exon 1 of the K-ras gene were detected in two of 13 human endometrial carcinoma [15%]; both were histologically defined adenocarcinomas, stage Ib and stage IIa according to the FIGO. Alterations were also observed in the single case of endometrial metastasis of ovarian cancer. It is worthy of note that among the six women with hyperplasic endometrial lesions K-ras gene mutation were not reported. These data suggest that K-ras activation is rare in Polish women and when it does occur it is in cancerous, but not in precancerous, lesions of human endometrium.

[Expression of bcl-2 gene in glia-derived tumors of the brain]. / Ekspresja genu bcl-2 w glejopochodnych guzach mózgu.

Bcl-2 gene is an important factor in regulation of apoptosis as an inhibitor of this process. Its increased expression is observed in numerous neoplasms, what points out on importance of this gene in the process of oncogenesis. The aim of this paper was to assess the expression of bcl-2 gene in the cells of the glia-derived tumours of the brain. The method of Chomczynski and Sacchi with guanidine tiocyanate was applied to isolate a whole cellular RNA. Expression of bcl-2 gene was measured with a reverse transcription method by a synthesis of cDNA and amplification of gene fragment with specific oligonucleotides in polymerase chain reaction (RT-PCR). The expression of bcl-2 gene was found in cells of all examined glial tumours at the level of transcript. We carried out the discussion of the results and an attempt to explain the importance of bcl-2 gene expression of these neoplasms.

[Fibronectin gene expression in preneoplastic states and female genital neoplasms]. / Ekspresja genu fibronektyny w stanach przednowotworowych i nowotworach zenskich narzadów Plciowych.

In this paper the fibronectin gene expression level (Northern blotting) in 33 endometrial samples (residua post abortum, endometrium proliferativum, secretivum and involutivum, hyperplasia glandularis endometrii, adenocarcinoma endometrii) was measured and compared. Higher level of fibronectin gene expression level in endometrial hyperplasia and carcinoma than in other endometrial samples was found. Strong correlation between high fibronectin gene expression level in endometrial hyperplasia and carcinoma was found.

[The expression of c-myc gene detected by in situ hybridization in endometrial hyperplasia]. / Ekspresja protoonkogenu c-MYC wykazana metoda hybrydyzacji in situ w rozrostach gruczolakowatych endometrium.

The expression of c-MYC gene has been detected by using the method of in situ hybridization. It has been detected mainly in glandular cells but also in stromal cells in atypical endometrial hyperplasia in women after menopause regardless of their age. A similar expression has been found to occur in nonatypical case of hyperplasia in a women of premenopausal age.

[Expression of the survivin gene in normal and hyperplastic endometrium]. / Ekspresja genu surviviny w prawidlowym i rozrostowym endometrium.

OBJECTIVE: Surviving a novel anti-apoptotic gene is expressed during fetal development and in numerous human cancers. MATERIAL AND METHODS: We analyzed the expression of surviving in human normal endometrium: proliferative n = 8, secretive n = 4 cases and hyperplastic endometrium 6 cases. RESULTS: RT-PCR revealed surviving expression in all cases of normal and hyperplastic endometrium. The highest expression of surviving gene was observed in hyperplastic endometrium and in the late proliferative phase.

[Expression of the survivin gene in the scar endometriosis and in normal human endometrium]. / Ekspresja genu surviviny w tkance endometriozy w bliznie oraz w prawidlowym endometrium.

UNLABELLED: The survivin gene encoding a novel inhibitor of apoptosis protein (IAP) is located on chromosome (17q25). It is expressed during development and in human cancer in vivo. Endometriosis in cicatrix after cesarean section represents the only localization of endometriotic implant which etiology is known. It is believed that during the surgical procedure when the uterine cavity is open there is a high risk of decidualized endometrium implantation. The aim of the study was to estimate the expression of the survivin mRNA in the scar endometriosis after cesarean section (n = 6) and perineum endometriosis (n = 2) and in normal human endometrium (n = 12, reference group) using reverse transcribed-polymerase chain reaction (RT-PCR). METHODS: Total cellular RNA was isolated from the tissues using TriReagent according to the manufacturer's protocol. Total cellular mRNA was transcribed into cDNA with reverse transcriptase (RT) then the survivin gene fragment (430 bp) was amplified with PCR using specific primers. The products of the PCR reaction were separated on 2% agarose gel. The survivin gene was expressed in 4 of 6 cases of scar endometriosis after cesarean section and in 2/2 cases of perineum endometriosis. In all cases of normal endometrium we found the expression of survivin with a peak in the late proliferative phase. The antiapoptosis function of this gene product may play an important role in the endometriotic implant growth promotion.

[Gene K-ras mutation in cells of endometrial adenocarcinoma in women]. / Mutacje genu K-ras w komórkach gruczolakoraka endometrium kobiety.

A simple, sensitive method of DNA analysis of nucleotide substitutions, namely, single-strand conformation polymorphism analysis of polymerase chain reaction products (PCR-SSCP analysis) was used for detection of mutated K-ras gene in specimens of human endometrial cancer. The mobility shifts of single-strand DNA fragment was seen in 3 of 8 analysed neoplasms: two in stage I and one in stage II, according to FIGO classification, K-ras gene mutation was found in 3 out of 4 patients over 66 years old.

[The detection of CFTR gene mutation in patients with azoospermia]. / Identyfikacja mutacji genu CFTR u mezczyzn z azoospermia.

Cystic fibrosis (CF) is the most common autosomal recessive disorder in Caucasian population that involves the lungs, pancreas, sweat glands, intestine, liver and reproductive tract. The majority of men with CF are infertile due to a bilateral congenital absence of the vas deferens (CBAVD). The purpose of the research was to evaluate the occurrence frequency of alleles of CFTR gene in patients with azoospermia. Twenty four men from sterile marriages were examined in whom spermatozoa were not found in 2-3 successive examinations of their semen. In every patient the basic concentration in blood of FSH, LH, PRL and testosterone was determined and ultrasonography of gonads, vas deferens, prostate and spermatic vesicles was performed. The INNO-LIPA CF2 test was used to the screening for the eight most frequently identified mutations in Caucasian population: DF508, G542X, N1303K, 1717-1(G- > A), W1282X, G551D, R553X i DI507. The test is based on the reverse-hybridization method. Carrier-state of one mutated allele was detected in 3 men (12.5% examined causes). In two men DF508 mutation and in one W1282X mutation were detected. The data suggest that the CFTR protein may be involved in the process of spermatogenesis apart from playing a critical role in the development of the vas deferens.

[P21-ras protein in tumors of the large intestine]. / Bialko p21-ras w rakach jelita grubego.

We carried out a prospective analysis of cytoplasmatic accumulation of p21ras protein-a Ki-ras gene product. The study was completed on the group of 80 patients with sporadic colorectal cancer. p21ras protein was detected immunohistochemically with use of NCC-001 antibody. In 64/80 cases (80%) we found p21ras protein accumulation in the tumor. We found that the protein accumulation is present more often in poorly differentiated cancers 91.3% (42/46) than in well and intermediate differentiated 64.7% (22/34) and in the right side of the colon 100% (14/14) than in left side colon 53.3% (14/24). The differences were statistically significant (p < 0.05). We found no statistically significant differences in survival time after surgery in correlation with p21ras accumulation in tumor. Our result suggest that p21ras accumulation is not a solitary prognostic factor in sporadic colorectal cancer.

[Detection of human papillomavirus type 16 and 18 in laryngeal cancer using PCR methods]. / Wykrywanie obecnosci wirusa brodawczaka ludzkiego HPV 16 i 18 w raku krtani z zastosowaniem metody PCR.

Human papillomavirus (HPV) is a small DNA virus. HPV is can be divided into two groups: mucosal and cutaneous. HPV have various oncogenic potential. The correlation between presence of high oncogenic type of HPV and carcinogenesis was confirmed in human anogenital tract and cervical carcinoma. The latest studies suggest HPV infection involvement in benign and malignant lesions of aerodigestive tract. The aim of our study was to determine the presence of the HPV genes E6/E7. The specimens were taken after total laryngectomy from 20 patients with squamous cell carcinoma. The fresh tissue specimens were frozen at -70 degrees C until DNA extraction and HPV detection. The presence of HPV DNA was assessed by polymerase chain reaction using specific primers for HPV 16 and 18 E6/E7. There were 7 (35%) HPV positive specimens in our group. 5 (25%) specimens were HPV 16 positive, 4 (20%) specimens were HPV-18 positive. In two cases HPV 16 and HPV 18 was present. The results suggest that high oncogenic types of HPV may play a role in pathogenesis of laryngeal carcinoma.

Genetic polymorphism of alcohol-metabolizing enzyme and alcohol dependence in Polish men.

Alcohol dependence poses a serious medical and sociological problem. It is influenced by multiple environmental and genetic factors, which may determine differences in alcohol metabolism. Genetic polymorphism of the enzymes involved in alcohol metabolism is highly ethnically and race dependent. The purpose of this study was to investigate the differences, if present, in the allele and genotype frequency of alcohol dehydrogenase 1B (ADH1B), ADH1C and the microsomal ethanol-oxidizing system (MEOS/CYP2E1) between alcohol-dependent individuals and controls and also to determine if these genotypes cause a difference in the age at which the patients become alcohol dependent. The allele and genotype frequencies of ADH1B, ADH1C, and CYP2E1 were determined in 204 alcohol dependent men and 172 healthy volunteers who do not drink alcohol (control group). Genotyping was performed by PCR-RFLP methods on white cell DNA. ADH1B*1 (99.3%) and ADH1C*1 (62.5%) alleles and ADH1B*1/*1 (N = 201) and ADH1C*1/*1 (N = 85) genotypes were statistically more frequent among alcohol-dependent subjects than among controls (99.3 and 62.5%, N = 201 and 85 vs 94.5 and 40.7%, N = 153 and 32, respectively). Differences in the CYP2E1 allele and genotype distribution between groups were not significant. The persons with ADH1C*1/*1 and CYP2E1*c1/*c2 genotypes became alcohol dependent at a considerably younger age than the subjects with ADH1C*1/*2, ADH1C*2/*2 and CYP2E1*c1/*c1 genotypes (28.08, 25.67 years vs 36.0, 45.05, 34.45 years, respectively). In the Polish men examined, ADH1C*1 and ADH1B*1 alleles and ADH1C*1/*1 and ADH1B*1/*1 genotypes favor alcohol dependence. The ADH1B*2 allele may protect from alcohol dependence. However, subjects with ADH1C*1/*1 and CYP2E1*c1/*c2 genotypes become alcohol dependent at a considerably younger age than the subjects with ADH1C*1/*2, ADH1C*2/*2 and CYP2E1*c1/*c1 genotypes.