hTERT gene methylation in circulating DNA, tumor, and surrounding tissue in breast cancer: a prospective study.

BACKGROUND: The human telomerase reverse transcriptase (hTERT) enzyme, encoded by the hTERT gene, synthesizes protective telomeric sequences on chromosomes and plays a fundamental role in cancer formation. Methylation of the hTERT gene has an upregulatory effect, increasing hTERT enzyme synthesis and allowing continuous tumor cell division. OBJECTIVE: In a group of patients with breast cancer, we aimed to analyze the methylation status of hTERT in the tumor, surrounding tissue, and circulating free deoxyribonucleic acid (cfDNA) of blood collected on the day of mastectomy and then approximately one year later. DESIGN AND SETTING: A prospective study was conducted at a university hospital in Rio de Janeiro, Brazil. METHODS: Samples were collected from 15 women with breast cancer on the day of mastectomy and approximately one year postoperatively. cfDNA was analyzed by sodium bisulfite conversion, followed by polymerase chain reaction, electrophoresis, and silver nitrate staining. RESULTS: Methylation of hTERT was detected in the tumors and surrounding tissues of all 15 patients. Five patients displayed hTERT methylation in the cfDNA from the blood of the first collection. Of the ten patients who returned for the second collection, three showed methylation. Two patients with methylation in the first collection did not display methylation in the second collection. One patient with no methylation in the first collection displayed methylation in the second collection, and one patient had a diminished level of methylation in the second collection. CONCLUSION: Only one-third of patients displayed methylation in their cfDNA, which may be related to the success of chemotherapy.

Molecular monitoring by CDKN2A/p16INK4A and RB1 gene methylation in breast cancer.

OBJECTIVE: This prospective study aimed to provide a comprehensive analysis of the methylation status of two pivotal genes, CDKN2A/p16INK4A (cyclin-dependent kinase inhibitor 2A) and RB1 (retinoblastoma transcriptional corepressor 1), in breast cancer patients. METHODS: Samples were obtained from 15 women diagnosed with breast cancer and who underwent a total mastectomy. DNA was extracted from the tumor, non-tumor tissue, and peripheral blood (circulating cell-free DNA). The methylation pattern of cell-free DNA extracted from blood collected on the day of mastectomy was compared with the methylation pattern of cell-free DNA from blood collected 1 year post-surgery. The methylation analysis was carried out by sodium bisulfite conversion and polymerase chain reaction, followed by electrophoresis. RESULTS: Methylation of CDKN2A/p16INK4A was identified in 13 tumor samples and 12 non-tumor tissue samples. Two patients exhibited CDKN2A/p16INK4A methylation in the cell-free DNA of the first blood collection, while another showed methylation only in the cell-free DNA of the subsequent blood collection. Regarding RB1, 11 tumors and 8 non-tumor tissue samples presented methylation of the gene. CONCLUSION: This study presents a novel approach for monitoring breast cancer patients through the analysis of cell-free DNA methylation. This analysis can detect changes in methylation patterns before any visible sign of cancer appears in breast tissue and could help predict the recurrence of malignant breast tumors.

Molecular monitoring by CDKN2A/p16INK4A and RB1 gene methylation in breast cancer

SUMMARY OBJECTIVE: This prospective study aimed to provide a comprehensive analysis of the methylation status of two pivotal genes, CDKN2A/p16INK4A (cyclin-dependent kinase inhibitor 2A) and RB1 (retinoblastoma transcriptional corepressor 1), in breast cancer patients. METHODS: Samples were obtained from 15 women diagnosed with breast cancer and who underwent a total mastectomy. DNA was extracted from the tumor, non-tumor tissue, and peripheral blood (circulating cell-free DNA). The methylation pattern of cell-free DNA extracted from blood collected on the day of mastectomy was compared with the methylation pattern of cell-free DNA from blood collected 1 year post-surgery. The methylation analysis was carried out by sodium bisulfite conversion and polymerase chain reaction, followed by electrophoresis. RESULTS: Methylation of CDKN2A/p16INK4A was identified in 13 tumor samples and 12 non-tumor tissue samples. Two patients exhibited CDKN2A/p16INK4A methylation in the cell-free DNA of the first blood collection, while another showed methylation only in the cell-free DNA of the subsequent blood collection. Regarding RB1, 11 tumors and 8 non-tumor tissue samples presented methylation of the gene. CONCLUSION: This study presents a novel approach for monitoring breast cancer patients through the analysis of cell-free DNA methylation. This analysis can detect changes in methylation patterns before any visible sign of cancer appears in breast tissue and could help predict the recurrence of malignant breast tumors.

hTERT gene methylation in circulating DNA, tumor, and surrounding tissue in breast cancer: a prospective study

ABSTRACT BACKGROUND: The human telomerase reverse transcriptase (hTERT) enzyme, encoded by the hTERT gene, synthesizes protective telomeric sequences on chromosomes and plays a fundamental role in cancer formation. Methylation of the hTERT gene has an upregulatory effect, increasing hTERT enzyme synthesis and allowing continuous tumor cell division. OBJECTIVE: In a group of patients with breast cancer, we aimed to analyze the methylation status of hTERT in the tumor, surrounding tissue, and circulating free deoxyribonucleic acid (cfDNA) of blood collected on the day of mastectomy and then approximately one year later. DESIGN AND SETTING: A prospective study was conducted at a university hospital in Rio de Janeiro, Brazil. METHODS: Samples were collected from 15 women with breast cancer on the day of mastectomy and approximately one year postoperatively. cfDNA was analyzed by sodium bisulfite conversion, followed by polymerase chain reaction, electrophoresis, and silver nitrate staining. RESULTS: Methylation of hTERT was detected in the tumors and surrounding tissues of all 15 patients. Five patients displayed hTERT methylation in the cfDNA from the blood of the first collection. Of the ten patients who returned for the second collection, three showed methylation. Two patients with methylation in the first collection did not display methylation in the second collection. One patient with no methylation in the first collection displayed methylation in the second collection, and one patient had a diminished level of methylation in the second collection. CONCLUSION: Only one-third of patients displayed methylation in their cfDNA, which may be related to the success of chemotherapy.

Epstein-Barr virus in gastric cancer and association with 30 bp del-latent membrane protein 1 polymorphism.

OBJECTIVE: This study aimed to determine the frequencies of Epstein-Barr virus, types 1 and 2 infection, and 30 bp del-latent membrane protein 1 viral polymorphism in gastric adenocarcinomas, as well as to investigate the association between Epstein-Barr virus infection and tumor location, type, and the patient's sex. METHODS: Samples were collected from 38 patients treated at a university hospital in Rio de Janeiro, Brazil. Epstein-Barr virus detection and genotyping were performed by polymerase chain reaction, followed by polyacrylamide gel electrophoresis and staining by the silver nitrate method. RESULTS: Overall, 68.4% of patients had Epstein-Barr virus-positive tumors. Of these, 65.4% presented infection by Epstein-Barr virus type 1, 23.1% by Epstein-Barr virus type 2, and 11.5% had coinfection with types 1 and 2. The 30 bp del-latent membrane protein 1 polymorphism was found in 42.3% of Epstein-Barr virus-positive tumors, 23.1% had the wild-type virus, and 23.1% had the wild-type and the polymorphism concomitantly. In 11.5% of Epstein-Barr virus-positive tumors, it was impossible to determine whether there was polymorphism or not. Tumor location in the antrum (22 of 38) and diffuse type (27 of 38) were predominant. There was no significant difference in Epstein-Barr virus infection or the 30 bp del-latent membrane protein 1 polymorphism between men and women. CONCLUSION: Epstein-Barr virus infection was found in 68.4% of tumors investigated in this study. To the best of our knowledge, this is the first article showing the coinfection of Epstein-Barr virus types 1 and 2 in gastric carcinoma in Brazil.

Epstein-Barr virus in gastric cancer and association with 30 bp del-latent membrane protein 1 polymorphism

SUMMARY OBJECTIVE: This study aimed to determine the frequencies of Epstein-Barr virus, types 1 and 2 infection, and 30 bp del-latent membrane protein 1 viral polymorphism in gastric adenocarcinomas, as well as to investigate the association between Epstein-Barr virus infection and tumor location, type, and the patient's sex. METHODS: Samples were collected from 38 patients treated at a university hospital in Rio de Janeiro, Brazil. Epstein-Barr virus detection and genotyping were performed by polymerase chain reaction, followed by polyacrylamide gel electrophoresis and staining by the silver nitrate method. RESULTS: Overall, 68.4% of patients had Epstein-Barr virus-positive tumors. Of these, 65.4% presented infection by Epstein-Barr virus type 1, 23.1% by Epstein-Barr virus type 2, and 11.5% had coinfection with types 1 and 2. The 30 bp del-latent membrane protein 1 polymorphism was found in 42.3% of Epstein-Barr virus-positive tumors, 23.1% had the wild-type virus, and 23.1% had the wild-type and the polymorphism concomitantly. In 11.5% of Epstein-Barr virus-positive tumors, it was impossible to determine whether there was polymorphism or not. Tumor location in the antrum (22 of 38) and diffuse type (27 of 38) were predominant. There was no significant difference in Epstein-Barr virus infection or the 30 bp del-latent membrane protein 1 polymorphism between men and women. CONCLUSION: Epstein-Barr virus infection was found in 68.4% of tumors investigated in this study. To the best of our knowledge, this is the first article showing the coinfection of Epstein-Barr virus types 1 and 2 in gastric carcinoma in Brazil.

JC and BK virus DNA detection in archival slides of urine cytospin from renal transplant patients.

BACKGROUND: Although identifying cytological viral inclusions (decoy cells) in the urine is relatively easy, distinguishing between Polyomaviruses BKV and JCV is not possible. Few studies have been published regarding JCV detection in kidney transplant recipients. OBJECTIVE: To evaluate the incidence of BKV and JCV DNA in archival slides of urine cytospin material from renal transplant patients. METHODS: A total of 44 urine specimens were evaluated cytologically for the presence of viral inclusions (decoy cells) and by nested polymerase chain reaction to differentiate between JCV and BKV in DNA isolated from archival slides of urine cytospin material. RESULTS: Of the 44 urine specimen donors, 9 (20.5%) patients had at least 1 sample with alterations suggestive of or compatible with viral infection (decoy cells), and 3 had urine samples with cellular atypias/neoplasias. Additionally, 24/44 (54.5%) patients had PCR-positive DNA for Polyomavirus in at least 1 sample, including 11/44 who were positive for BKV (25%) and 16/44 who were positive for JCV (36.36%), with 3 (6.8%) patients showing viral coinfection. Regarding transplantation time, only JCV was statistically significant (P = .019) for periods longer than 10 years. CONCLUSIONS: The results highlight the potential use of archival slides of urine cytospin material to differentiate BKV and JCV and demonstrate the importance of improved JCV detection for later kidney transplant recipients.

Green Tobacco Sickness among Brazilian farm workers and genetic polymorphisms.

OBJECTIVE: Green Tobacco Sickness (GTS) is an occupational illness caused by dermal absorption of nicotine from tobacco leaves. It affects thousands of farm workers worldwide. Brazil is the second tobacco producer in the world; despite this, there are few studies on GTS among Brazilian harvesters. This study aimed to determine the prevalence of GTS among a population of tobacco workers from a producing area in northeastern Brazil and investigate whether the occurrence of the disease was influenced by factors such age, gender and smoking status. In addition, it was investigated if there was association between the onset of GTS and genetic polymorphisms in genes that encode some detoxification enzymes. A semi-structured questionnaire was used to collect demographic, behavioral and occupational data from the referred workers. Polymorphisms were tested through the Polymerase Chain Reaction technique. RESULTS: The total prevalence of GTS found was 56.9%, with a significant difference between genders (71.7% for women and 35.3% for men, p < 0.0001). No association was identified between the investigated polymorphisms and GTS. This study confirms the occurrence of GTS among tobacco harvesters in Brazil with high prevalence. The investigation suggests the need to take preventive measures to protect tobacco workers against this disease.

Colorectal cancer DNA methylation patterns from patients in Manaus, Brazil.

BACKGROUND: DNA methylation is commonly linked with the silencing of the gene expression for many tumor suppressor genes. As such, determining DNA methylation patterns should aid, in times to come, in the diagnosis and personal treatment for various types of cancers. Here, we analyzed the methylation pattern from five colorectal cancer patients from the Amazon state in Brazil for four tumor suppressor genes, viz.: DAPK, CDH1, CDKN2A, and TIMP2 by employing a polymerase chain reaction (PCR) specific to methylation. Efforts in the study of colorectal cancer are fundamental as it is the third most of highest incidence in the world. RESULTS: Tumor biopsies were methylated in 1/5 (20%), 2/5 (40%), 4/5 (80%), and 4/5 (80%) for CDH1, CDKN2A, DAPK, and TIMP2 genes, respectively. The margin biopsies were methylated in 3/7 (43%), 2/7 (28%), 7/7 (100%), and 6/7 (86%) for CDH1, CDKN2A, DAPK, and TIMP2, respectively. CONCLUSIONS: Our findings showed DAPK and TIMP2 to be methylated in most samples from both tumor tissues and adjacent non-neoplastic margins; thus presenting distinct methylation patterns. This emphasizes the importance of better understanding of the relation of these patterns with cancer in the context of different populations.

Colorectal cancer DNA methylation patterns from patients in Manaus, Brazil

BACKGROUND: DNA methylation is commonly linked with the silencing of the gene expression for many tumor suppressor genes. As such, determining DNA methylation patterns should aid, in times to come, in the diagnosis and personal treatment for various types of cancers. Here, we analyzed the methylation pattern from five colorectal cancer patients from the Amazon state in Brazil for four tumor suppressor genes, viz.: DAPK, CDH1, CDKN2A, and TIMP2 by employing a polymerase chain reaction (PCR) specific to methylation. Efforts in the study of colorectal cancer are fundamental as it is the third most of highest incidence in the world. RESULTS: Tumor biopsies were methylated in 1/5 (20 %), 2/5 (40 %), 4/5 (80 %), and 4/5 (80 %) for CDH1, CDKN2A, DAPK, and TIMP2 genes, respectively. The margin biopsies were methylated in 3/7 (43 %), 2/7 (28 %), 7/7 (100 %), and 6/7 (86 %) for CDH1, CDKN2A, DAPK, and TIMP2, respectively. CONCLUSIONS: Our findings showed DAPK and TIMP2 to be methylated in most samples from both tumor tissues and adjacent non-neoplastic margins; thus presenting distinct methylation patterns. This emphasizes the importance of better understanding of the relation of these patterns with cancer in the context of different populations.

Teste de biocompatibilidade e resistência de membranas de látex em cães / Biocompatibility and resistance test of latex membranes in dogs

O látex natural extraído da seringueira (Hevea brasiliensis) possui propriedades indutoras de neovascularização e regeneração tecidual, comprovadas em várias espécies e em diferentes tecidos do organismo. Este estudo testou a biocompatibilidade e a resistência de três membranas de látex em seis cadelas, sendo duas dessas membranas ainda não utilizadas e uma já testada em estudos prévios. Os implantes foram colocados entre os músculos cutâneo e reto do abdome através de procedimento cirúrgico e, após 45 dias, foram removidos e submetidos a exames histológicos. Durante esse período, os animais foram avaliados quanto à dor e às alterações clínicas e macroscópicas nas membranas. Testes mecânicos de tração para determinação de deformação e resistência foram efetuados em amostras-controle. Pelos resultados obtidos, concluiu-se que as membranas testadas são compatíveis para substituir a bainha muscular em cães, exceto a membrana 2, por apresentar características de rejeição.

The natural latex extracted of the Rubber Tree, has properties to induce characteristics of tissue vascularization and regeneration that had been comproved in different species and tissues. This study tested the biocompatibility of three latex membranes in six dogs. Two of these membranes have not been used yet, while one of them has been tested in previous studies. Membranes were implanted between the cutaneous and the rectum of the abdomen muscles by a surgical procedure. In a forty five days period, the animals were evaluated for pain, clinical and macroscopic alterations of the membranes and, after the membranes were removed in order to submit them to histological exams. Mechanical traction tests were realized in control membranes to measure deformation and resistance. The results indicated that membranes are compatible, and able to substitute the muscular sheath in dogs, excepting membrane 2, because of it rejecting characteristics.

Effects of perillyl alcohol and heat shock treatment in gene expression of human lung adenocarcinoma cell line A549.

The K-Ras protein is found mutated in 42.4% of lung adenocarcinoma cases, evidencing its importance as a chemotherapeutic target. The Ras protein becomes functional after farnesylation, a post-transduction modification, allowing its attachment to the cellular membrane permitting signal transduction. Perillyl alcohol (POH) has been shown to inhibit the farnesylation of small G-proteins such as Ras. HSP70, a protein known to appear after heat shock (HS), is found over expressed in lung cancer and modifies chemotherapeutic effects. In this work, the effect of POH and HS in the gene expression of human adenocarcinoma lung cells (A549) is studied. Cells incubated with POH followed by 42 degrees C HS presented a 20.7% cellular viability decrease compared to the ones kept at 37 degrees C. A different pattern synthesis was observed for each sequences of cell treatment. Independent of the heat treatment, the amount of HSP70 was decrease by POH without modification in the amount of p53. Here it is shown that HS modified the POH effects in the ERK activation pathway by altering the phosphorylation of p44/42 in human adenocarcinoma lung cells.

Efeito do álcool perílico na expressão gênica de células de adenocarcinoma de pulmão humano / Effect of perillyl alcohol on gene expression of human pulmonary adenocarcinoma cells

OBJETIVO: Estudar a ação do álcool perílico na expressão gênica de células de adenocarcinoma de pulmão humano. MÉTODOS: Incubaram-se células de adenocarcinoma de pulmão com álcool perílico em diluições que variaram entre 0,03 por cento e 0,0003 por cento por 48 horas. Observaram-se as alterações na morfologia celular e quantificou-se a viabilidade celular pelo método do MTT (3-(4,5-dimetiltiazol-2-yl)-2,5 difeniltertrazolim brometo). Analisou-se a síntese de proteínas das amostras previamente marcadas radioativamente com 35S, através de eletroforese em gel de poliacrilamida. Determinou-se a expressão das proteínas p53 e p42/44 através do método de Western Blot. RESULTADOS: Após 48 horas de incubação, observaram-se alterações na morfologia celular para a diluição de 0,03 por cento de álcool perílico, as quais foram pouco verificadas em diluições superiores a 0,003 por cento. A inibição da viabilidade celular foi de 60,17 por cento (p < 0,001), 15,62 por cento (p < 0,001) e 11,53 por cento (p < 0,05) para as diluições de 0,03 por cento, 0,003 por cento e 0,0003 por cento de álcool perílico, respectivamente. Os resultados mostram a indução de proteínas de 110 kDa, 42 kDa e 28 kDa. Não se observou variação estatisticamente significativa para a expressão da proteína p53. Em comparação com a expressão de alfa-tubulina, a diluição de 0,003 por cento de álcool perílico provocou uma diminuição marcante da fosforilação da p44 e um aumento da fosforilação da p42. CONCLUSÃO: Os resultados apresentados sugerem novos caminhos metabólicos da ação do álcool perílico em células de adenocarcinoma de pulmão humano.

OBJECTIVE: To study the effect of perillyl alcohol on the gene expression of human pulmonary adenocarcinoma cells. METHODS: Pulmonary adenocarcinoma cells were incubated with perillyl alcohol in dilutions ranging from 0.03 percent to 0.0003 percent for 48 hours. Alterations were observed in the cell morphology, and cell viability was quantified using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays. Protein synthesis of samples previously targeted with S35 was analyzed using electrophoresis on a polyacrylamide gel. Expression of the proteins p53 and p44/42 was determined using the Western blot method. RESULTS: After 48 hours of incubation, greater nsumbers of morphological alterations were observed in cells treated with the 0.03 percent perillyl alcohol dilution than in those treated with perillyl alcohol diluted to 0.003 percent or further. Treatment with perillyl alcohol dilutions of 0.03 percent, 0.003 percent and 0.0003 percent inhibited cellular viability by 60.17 percent (p < 0.001), 15.62 percent (p < 0.001) and 11.53 percent (p < 0.05), respectively. The results show that 28-kDa, 42-kDa and 110-kDa proteins were induced. No statistically significant effect on p53 expression was observed. In comparison with the expression of alpha-tubulin, the 0.003 percent perillyl alcohol dilution induced an increase in p42 phosphorylation and a marked decrease in p44 phosphorylation. CONCLUSION: The results suggest that there are other, previously undescribed, metabolic pathways for perillyl alcohol effects in human pulmonary adenocarcinoma cells.

Detecção de potenciais marcadores moleculares séricos da doença de Hodgkin / Detection of potential serum molecular markers for Hodgkin's disease

MOTIVAÇAO: Neste trabalho foi analisado o perfil de proteínas séricas de pacientes com doença de Hodgkin (DH) localizada e avançada em busca de novos e potenciais biomarcadores para o diagnóstico médico. MATERIAIS E MÉTODOS: O perfil de proteínas presentes no soro de 14 indivíduos saudáveis, 14 pacientes com DH avançada e 15 pacientes com DH localizada, assim como pools de soro dos respectivos grupos, foi analisado em gel desnaturante de poliacrilamida a 12 por cento corado pela prata. A densitometria e a intensidade média das bandas de interesse foram estudadas utilizando-se o Kodak 1D Scientific Imaging System. RESULTADOS E CONCLUSÕES: O perfil protéico apresentou acentuada variação entre os pacientes examinados; entretanto foi observada a indução predominante de determinadas proteínas (aproximadamente 26kDa e 18kDa), cuja expressão foi substancialmente diferente quando em comparação com os controles (p < 0,01). Estas proteínas podem potencialmente constituir-se em marcadores moleculares de acompanhamento da evolução e do tratamento da doença.