RESUMO
We evaluated the prognostic significance of p185c-erbB-2 expression and ras gene mutations in all patients diagnosed with a pulmonary adenocarcinoma between 1982 and 1985 at the University of Iowa. p185c-erbB-2 expression was detected in 15 cases (34%). A ras gene mutation was found in 16 cases (36%) and all were in codon-12 of K-ras. No N-ras mutations were identified. Both p185c-erbB-2 expression and a K-ras mutation were found only in codon-12 and present in six cases (14%). By univariate analysis p185c-erbB-2 expression was associated with shortened survival (P = 0.02) while the presence of a K-ras mutation was not (P = 0.16). Multivariate analysis by the Cox proportional hazards model, controlling for patient age and tumor stage, also continued to identify p185c-erbB-2 expression as an independent unfavorable prognostic factor (P = 0.01). In this model a K-ras mutation also approached significance as a poor prognostic indicator (P = 0.06). The impact of both p185c-erbB-2 expression and a K-ras mutation on survival was additive and highly significant (P = 0.004). This additive nature suggests that together these two markers identify a high-risk population of lung adenocarcinoma patients that may benefit from aggressive therapy.
Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/análise , Receptores ErbB/biossíntese , Genes ras , Neoplasias Pulmonares/genética , Mutação Puntual , Proteínas Proto-Oncogênicas/biossíntese , Proto-Oncogenes , Análise Atuarial , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Fatores Etários , Sequência de Bases , Primers do DNA , DNA de Neoplasias/isolamento & purificação , DNA de Neoplasias/metabolismo , Receptores ErbB/análise , Feminino , Humanos , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Dados de Sequência Molecular , Análise Multivariada , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase , Prognóstico , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas/análise , Receptor ErbB-2 , Fatores de Risco , Análise de Sobrevida , Fatores de TempoRESUMO
A mutation of the LDL receptor gene very common among Finnish patients with heterozygous familial hypercholesterolemia (FH) was identified. This mutation, designated as FH-North Karelia, deletes seven nucleotides from exon 6 of the LDL receptor gene, causes a translational frameshift, and is predicted to result in a truncated receptor protein. Only minute quantities of mRNA corresponding to the deleted gene were detected. Functional studies using cultured fibroblasts from the patients revealed that the FH-North Karelia gene is associated with a receptor-negative (or binding-defective) phenotype of FH. Carriers of the FH-North Karelia gene showed a typical xanthomatous form of FH, with mean serum total and LDL cholesterol levels of 12 and 10 mmol/liter, respectively. This mutation was found in 69 (34%) out of 201 nonrelated Finnish FH patients and was especially abundant (prevalence 79%) in patients from the eastern Finland. These results, combined with our earlier data on another LDL receptor gene deletion (FH-Helsinki), demonstrate that two "Finnish-type" mutant LDL receptor genes make up about two thirds of FH mutations in this country, reflecting a founder gene effect. This background provides good possibilities to examine whether genetic heterogeneity affects the clinical presentation or responsiveness to therapeutic interventions in FH.
Assuntos
Deleção Cromossômica , Éxons , Hiperlipoproteinemia Tipo II/genética , Mutação , Receptores de LDL/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , Finlândia , Humanos , Hiperlipoproteinemia Tipo II/etiologia , Lipídeos/sangue , Dados de Sequência Molecular , Fenótipo , RNA Mensageiro/análiseRESUMO
We have studied the effect of in vivo treatment with trioxsalen on DNA replication in mammalian cells. In vitro cultured bovine liver cells were exposed to two or four cycles of treatment with 45 microM trioxsalen followed by irradiation with long-wave ultraviolet light. Thymidine incorporation was reduced by about 95% during the first hour after a double treatment. A large proportion of the label was released in alkaline sucrose gradients as a low molecular weight fraction (average length about 500 nucleotides) which was supposed to consist of replication origins containing DNA fragments. From the relative quantities of this DNA obtained at different times of the S phase we concluded that it contains a considerable but not precisely determinable proportion of non-origin DNA. We also find that the fraction is contaminated by a large excess of non-replicating bulk DNA.
Assuntos
Replicação do DNA , DNA/biossíntese , Furocumarinas/farmacologia , Trioxsaleno/farmacologia , Animais , Bovinos , Fracionamento Celular/métodos , Linhagem Celular , DNA/isolamento & purificação , Replicação do DNA/efeitos dos fármacos , Timidina/metabolismoRESUMO
PURPOSE: To determine whether the clinical course and the response to chemotherapy of patients with advanced adenocarcinoma of the lung depends on the presence or absence of a ras mutation in the tumor. Mutational activation of K-ras is a strong adverse prognostic factor in stage I or II lung cancer and laboratory studies have suggested that ras mutations lead to resistance against ionizing radiation and chemotherapy. PATIENTS AND METHODS: Patients with advanced adenocarcinoma of the lung with measurable or assessable disease received chemotherapy with mesna, ifosfamide, carboplatin, and etoposide (MICE). Archival biopsies, fresh biopsies, or fine-needle aspirations were tested for the presence of ras gene mutations. Associations of ras mutations with clinical characteristics, response to chemotherapy, and survival were studied. RESULTS: The presence or absence of ras gene mutations could be established in 69 of 83 patients (83%). A total of 261 courses of MICE were administered to 62 informative patients, 16 of whom were shown to have a K-ras mutation-positive tumor. The frequency of mutations (26%) and the type of mutations closely matched the pattern we have previously reported in operable disease. Patients with a ras mutation in their tumor were more likely to have a close relative with lung cancer, but other clinical characteristics, such as pattern of metastases, response, and survival, were similar between the ras mutation-positive and ras mutation-negative groups. CONCLUSION: Patients with advanced lung adenocarcinoma who harbor a ras mutation may have major responses to chemotherapy and have similar progression-free and overall survival as patients with ras mutation-negative tumors. K-ras mutations may represent one of several ways in which early tumors are enabled to metastasize to distant sites.
Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes ras/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação/genética , Adulto , Idoso , Carboplatina/administração & dosagem , Etoposídeo/administração & dosagem , Feminino , Humanos , Ifosfamida/administração & dosagem , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Análise de SobrevidaRESUMO
DNA samples from 53 unrelated Dutch patients with familial hypercholesterolemia (FH) were screened for rearrangements in the gene for the LDL receptor (LDLR) by Southern analysis. Four different mutations have been detected by hybridisation of BglII digested genomic DNA with an exon 10-14 containing cDNA probe. The mutations are defined by a 7 kb insertion near exon 11, a partial gene duplication encompassing exons 9-12, a 4 kb deletion of exons 7 and 8 and an 0.4 kb deletion comprising the 5'-part of exon 16. These four different rearrangements in the LDLR gene account for 17% of the mutations in the Dutch FH population sample. Interestingly, the 4 kb deletion was detected in 5 unrelated FH patients (9.5%) and appeared to be identical to the deletion previously described (Russell, D.W. et al., Arteriosclerosis, 9 (Suppl. I) (1989) I-8; Russell, D.W. et al., Cold Spring Harbor Symp. Quant. Biol., 51 (1987) 401). in an FH patient of Dutch origin. This suggests that the 4 kb deletion is a common mutation in the Dutch FH population.
Assuntos
Deleção Cromossômica , Rearranjo Gênico/genética , Hiperlipoproteinemia Tipo II/genética , Receptores de LDL/genética , Mapeamento Cromossômico , Análise Mutacional de DNA , Sondas de DNA , Triagem de Portadores Genéticos , Humanos , Mutação/genética , Países Baixos , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de RestriçãoRESUMO
In this report, we describe the characterization of a mutation in the low density lipoprotein (LDL) receptor gene of a true homozygous familial hypercholesterolemic (FH) patient. The combined use of denaturing gradient gel electrophoresis (DGGE) and DNA sequence analysis revealed a unique A to G transition in the penultimate 3'-nucleotide of intron 16 of the LDL receptor gene, which disrupts the acceptor splice site. cDNA sequence analysis indicated that a cryptic splice site was activated in intron 16, upstream from the original splice site, leading to the inclusion of 62 nucleotides and a reading frame-shift. The resulting new translation product contains a stretch of 154 amino acids at the carboxy-terminal that have no resemblance to the normal receptor protein. To elucidate the biological effects of the mutation, the structural and functional properties of the mutated LDL receptor protein were studied. Immunoprecipitation of the newly synthesized LDL receptors showed that an aberrant precursor form of the LDL receptor protein was synthesized, about 10 kDa larger than normal, which is not further processed to the mature form. Some 50% of the normal LDL binding activity was found on the cell surface of the patient's fibroblasts, whereas internalization and degradation of LDL were abolished.
Assuntos
Hiperlipoproteinemia Tipo II/genética , Íntrons/genética , Mutação Puntual , Splicing de RNA , Receptores de LDL/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas , Eletroforese , Feminino , Homozigoto , Humanos , Hiperlipoproteinemia Tipo II/metabolismo , Dados de Sequência Molecular , Linhagem , Fases de Leitura , Receptores de LDL/metabolismo , Análise de Sequência de DNARESUMO
BACKGROUND: Human cytomegalovirus (CMV) infection is a major cause of morbidity in transplant patients. Early diagnosis and treatment have been shown to improve outcome. We evaluated the suitability of CMV immediate early, early, and late gene expression detected by nucleic acid sequence-based amplification (NASBA) as markers of CMV infection. METHODS: Blood samples were taken immediately before transplant and every one to two weeks after transplantation for 12 weeks from 50 patients undergoing thoracic organ transplantation. CMV-NASBA was performed and results compared with serology, CMV pp65 antigenaemia (CMV-AG) and the development of clinical CMV infection. Patients received "preemptive" anti-CMV therapy with ganciclovir based on the CMV-AG results. RESULTS: CMV immediate early and early gene expression were detected in 87 and 47%, respectively, of patients without other evidence of CMV infection. CMV late gene expression had a sensitivity of 97% for infection (compared with 83% for CMV-AG P=0.06) and a specificity of 93% (compared with 100% P=NS). Late gene expression occurred at the same time as CMV antigenaemia but 1.1 weeks earlier than the threshold of antigenaemia (CMV-AG>10) used to initiate preemptive therapy. CONCLUSION: NASBA provided a standardized tool for the detection of CMV transcripts with a greater sensitivity than the standard antigenemia test. Detection of immediate early and early gene transcripts was not specific for subsequent infection. CMV late gene expression determined by NASBA was an accurate and early marker of CMV infection. Detection of CMV late gene expression could be used to trigger "preemptive" anti-CMV therapy.
Assuntos
Citomegalovirus/genética , Genes Virais/genética , Transplante de Coração , Transplante de Coração-Pulmão , Transplante de Pulmão , Replicação de Sequência Autossustentável/métodos , Adulto , Antivirais/uso terapêutico , Infecções por Citomegalovirus/sangue , Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Citomegalovirus/imunologia , Feminino , Ganciclovir/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Transcrição GênicaRESUMO
To find early and sensitive indicators of treatment response in breast cancer, we studied the mRNA levels of proliferation-related genes during growth arrest of the human breast cancer cell lines T47D and MCF7. A sensitive reverse transcriptase-PCR (RT-PCR) technique was used in order to monitor gene expression in small samples of cells. Estrogen-depletion and treatment with tamoxifen effectively induced a G1-arrest in both cell lines, accompanied by a decrease of the mRNA levels of histone H4, cyclin A, cyclin D1, and c-myc. Cyclin A expression decreased most strongly: up to 32-fold within 7 days. The expression of c-fos and WAF1 increased during growth arrest. In conclusion, significant changes of the levels of proliferation-related mRNAs, induced by growth arrest, can be measured in small samples of breast carcinoma cells using RT-PCR. Especially the decrease of the cyclin A mRNA level seems a potential early indicator of clinical response to tamoxifen therapy in breast cancer patients.
Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/metabolismo , Sequência de Bases , Neoplasias da Mama/patologia , Ciclo Celular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Primers do DNA/química , Feminino , Genes fos , Genes myc , Histonas/genética , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , RNA Neoplásico/metabolismo , Tamoxifeno/farmacologia , Células Tumorais CultivadasRESUMO
A method based on the reverse transcriptase-polymerase chain reaction (RT-PCR) was developed that allows the determination of relative mRNA expression levels in fine-needle aspirates from human tumors. The method was developed for the c-erbB-2 gene, using the porphobilinogen deaminase (PBGD) gene as an internal standard. It was validated for mRNA isolated from cell lines and for material obtained by fine-needle aspiration from human breast cancer. Gene expression levels were determined by measuring the activity of radiolabeled RT-PCR-amplified gene-specific bands with a phosphor imager. At least four points are measured on the log-linear part of the amplification cycle versus signal intensity curves, and subsequently the distance between the curves of the gene of interest and that of an internal standard gene is used to calculate the relative expression levels. The method worked equally well with the BRCA1 gene, illustrating that it can be generalized to other genes. The method is suitable to measure or monitor semiquantitively gene expression levels in accessible human tumors in situ.
Assuntos
Neoplasias da Mama/química , Neoplasias da Mama/genética , RNA Mensageiro/análise , Proteína BRCA1/genética , Biópsia por Agulha , Northern Blotting , Estudos de Viabilidade , Feminino , Expressão Gênica , Humanos , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/biossíntese , Análise de Regressão , Reprodutibilidade dos Testes , Células Tumorais CultivadasRESUMO
Nucleic Acid Sequence Based Amplification (iNASBA), an isothermal amplification technique for nucleic acids, was evaluated for the identification of medically important Candida species using primers selected from 18S rRNA sequences conserved in fungi. An RNA fragment of 257 nucleotides was amplified for Candida albicans. Nineteen different fungi were tested for rRNA amplification with the NASBA. All were positive when analyzed on agarose gel, whereas human RNA was negative. For the identification of Candida species, NASBA amplification products were analyzed in an enzyme bead-based detection format, using species-specific biotinylated probes and a generic Candida HRPO probe or a membrane-based system using biotinylated probes and avidin-HPRO. Discrimination of the major human pathogenic Candida spp. was based on a panel of biotinylated probes for C. krusei, C. tropicalis, C. albicans, C. glabrata, and C. lusitaniae. Using rRNA dilutions obtained from pure cultures of C. albicans, the combination of NASBA and the enzymatic bead-based detection yielded a sensitivity equivalent to 0.01 CFU. In a model system using 1 ml of artificially contaminated blood as few as 1-10 CFU of C. albicans could be detected. Testing of 68 clinical blood samples from patients suspected of candidemia showed that eight samples were positive for C. albicans and one for C. glabrata. Testing of 13 clinical plasma samples from patients suspected of fungemia identified the presence of C. albicans in two specimens. The whole procedure of sample preparation, amplification and identification by hybridization can be performed in 1 day. This speed and the observed sensitivity of the assay make the NASBA a good alternative to PCR for the detection of candidemia.
Assuntos
Candida/química , Candidíase/microbiologia , Fungemia/microbiologia , RNA Fúngico/análise , Humanos , Técnicas de Tipagem Micológica , Reação em Cadeia da Polimerase , RNA Antissenso , RNA Ribossômico 18S , Sensibilidade e EspecificidadeRESUMO
Human CMV (HCMV)-directed preemptive therapy has helped to improve the outcome following allo-SCT. In this study, we evaluated the safety and efficacy of a late mRNA-based (NucliSens CMV pp67) anti-HCMV treatment strategy. A prospective randomized multicenter pilot trial was performed comparing PCR-based, with late mRNA-based preemptive HCMV-directed antiviral therapy in patients after allo-SCT. In all, 133 patients were randomized in three different centers at the time of transplant, 130 of whom are evaluable. Viral screening was performed weekly. Antiviral therapy was initiated at the second consecutive positive PCR result, or at the first detection of late mRNA. The therapy was stopped if clearance of HCMV DNA or late mRNA was demonstrated after 14 days of antiviral therapy. If HCMV infection persisted, antiviral therapy was continued in a reduced dose. The median duration of antiviral therapy during the first treatment episode was 28 days for PCR-, and 19 days for mRNA-screened patients (P<0.02). However, the overall duration of antiviral therapy, as well as the incidence of HCMV disease and the OS at day 100 after transplantation was comparable between the two study groups. We conclude that late mRNA-based anti-HCMV therapy may show comparable safety and efficacy with PCR-based therapy in patients after allo-SCT.
Assuntos
Antivirais/administração & dosagem , Infecções por Citomegalovirus/etiologia , Infecções por Citomegalovirus/terapia , Citomegalovirus/isolamento & purificação , Fosfoproteínas/genética , RNA Mensageiro/sangue , Transplante de Células-Tronco , Proteínas da Matriz Viral/genética , Adolescente , Adulto , Criança , Pré-Escolar , Citomegalovirus/genética , Infecções por Citomegalovirus/sangue , Infecções por Citomegalovirus/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Estudos Prospectivos , RNA Mensageiro/genética , Transplante Homólogo , Resultado do Tratamento , Adulto JovemRESUMO
NASBA is an isothermal nucleic acid amplification reaction that amplifies mRNA in a dsDNA background. Although similar to the sensitive reverse transcription/polymerase chain reaction (RT-PCR) in mRNA detection, NASBA is not prone to give false positive results caused by genomic dsDNA. Therefore, NASBA is unique for sensitive detection of transcription of intronless genes, which preclude strategies such as intron spanning primer pairs to control false positive results in RT-PCR. Using NASBA, mRNA of the intronless human interferon-beta gene was demonstrated with a sensitivity of 10 copies, whereas 100 ng genomic DNA gave a negative result.
Assuntos
Expressão Gênica , Interferon beta/genética , Íntrons , Técnicas de Amplificação de Ácido Nucleico , RNA Mensageiro/isolamento & purificação , Reações Falso-Positivas , Humanos , Sensibilidade e EspecificidadeRESUMO
Microdissection of the lampbrush loops "threads" and "pseudonucleolus" of Y chromosomes from primary spermatocytes of Drosophila hydei and subsequent microcloning of the DNA yielded several recombinant DNA clones which cross-hybridized in screening the different clone banks. By DNA sequencing we found that the inserts of these cross-hybridizing clones contain blocks of poly[d(C-A].poly[d(G-T)]. Testis RNA contains a large fraction of transcripts with this simple repeated nucleotide sequence. With the aid of transcript in situ hybridization we discovered that the "cones" and "pseudonucleolus" lampbrush loops are the primary sites of transcription of poly[d(C-A)].poly[d(G-T)] in spermatocytes. In addition, we found a strand-specific transcription of (CA/GT)n. In both the "cones" and "pseudonucleolus" the (CA)n strand is transcribed, while in the "pseudonucleolus" (GT)n is also transcribed. Labelled (CA)n probes also react with the protein bodies in spermatid nuclei. These observations are discussed in the context of possible functions of (CA/GT)n transcripts in spermatogenesis.
Assuntos
Drosophila/genética , Polidesoxirribonucleotídeos/genética , Espermatócitos/metabolismo , Transcrição Gênica , Cromossomo Y , Animais , Sequência de Bases , Clonagem Molecular , Drosophila melanogaster/genética , Expressão Gênica , Masculino , Dados de Sequência Molecular , Mapeamento de Nucleotídeos , Sequências Repetitivas de Ácido Nucleico , Espermátides/metabolismo , TestículoRESUMO
We have examined 17 adenocarcinomas and 2 mixed tumors of the salivary glands for mutational activation of the oncogenes H-ras, K-ras and N-ras. The presence of mutations was determined by in vitro amplification of gene fragments spanning codons 12, 13 and 61 and the use of mutation-specific oligonucleotide hybridization. ras mutations were present in 3 of 13 adenocarcinomas (23%) of the parotid gland. The mutations were confirmed and characterized by means of cycle sequencing of polymerase chain reaction (PCR) fragments. In all 3 cases, the mutation was an A:T to G:C transition at the second position of codon 61 of the H-ras gene. This rather unusual ras mutation could provide a clue to identify one or more carcinogens involved in the pathogenesis of parotid cancer.
Assuntos
Adenocarcinoma/genética , Genes ras/genética , Neoplasias Parotídeas/genética , Mutação Puntual/genética , Sequência de Bases , Humanos , Dados de Sequência MolecularRESUMO
BACKGROUND: The heterogenous composition of tumors is a major obstacle for the measurement of mRNA levels in cancer cells. We report here a combination of immunomagnetic purification of cancer cells and reverse transcriptase polymerase chain reaction (RT-PCR) that enables highly sensitive detection of multidrug resistance gene 1 (MDR1)-mRNA levels in human breast carcinoma cells obtained from fine needle aspirates (FNA). EXPERIMENTAL DESIGN: Murine mAb 115D8 directed against episialin (MUC1/MAM6, epithelial membrane Ag) was used in combination with goat anti-mouse-coated magnetic microbeads to purify human T47D breast carcinoma cells (115D8+, MDR1-) from different mixtures with COLO320 human colon carcinoma cells (115D8-, MDR1+) and to purify carcinoma cells from FNA taken from axillary lymph node metastases in breast cancer patients. The efficacy of the purification was determined by FACS-analysis and by measurement of MDR1-mRNA levels by semiquantitative RT-PCR. RESULTS: FACS-analysis demonstrated that T47D cells could be purified up to 99.8% from mixtures with COLO320 cells ranging from 3:1 to 1:3. The MDR1-mRNA level in these enriched mixtures, as detected by RT-PCR, was reduced 250-fold. It was demonstrated that MDR1 expression present in an FNA from a lymph node metastasis of breast carcinoma could be attributed completely to the leukocytes present in this FNA, because MDR1 expression was no longer detectable after purification of the tumor cells. CONCLUSION: The combination of immunomagnetic purification of breast carcinoma cells and RT-PCR enables the measurement of cancer-specific MDR1 mRNA levels in small cell samples obtained by FNA.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Neoplasias da Mama/genética , Carcinoma/genética , Resistência a Múltiplos Medicamentos/genética , Separação Imunomagnética , RNA Mensageiro/metabolismo , Sequência de Bases , Biópsia por Agulha , Neoplasias da Mama/patologia , Carcinoma/patologia , Separação Celular/métodos , Neoplasias do Colo/patologia , Feminino , Humanos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/genética , Reação em Cadeia da Polimerase , DNA Polimerase Dirigida por RNA , Células Tumorais CultivadasRESUMO
Increasingly, reverse transcriptase polymerase chain reaction (RT-PCR) is used to detect clinically significant tumour cells in blood or bone marrow. This may result in a redefinition of disease-free and clinical relapse. However, its clinical utility may be limited by lack of automation or reproducibility. Recent studies have suggested nucleic acid sequence-based amplification of target RNA may be more robust. In this study, nucleic acid sequence-based amplification was established to detect melanoma, colorectal and prostate cancer cells. Nucleic acid sequence-based amplification and RT-PCR both successfully amplified target RNA in peripheral blood samples from patients with melanoma and colorectal cancer, but only RT-PCR detected PSA in blood samples from patients with prostate cancer. There was relatively good agreement between sample replicates analyzed by RT-PCR (Kappa values of one for tyrosinase, 0.67 for CK-20 and one for PSA), but less agreement when analyzed by nucleic acid sequence-based amplification. This may limit the routine use of NASBA for the detection of clinically significant disease. In summary, RT-PCR appears at present to be the most reliable and reproducible method for the detection of low-level disease in cancer patients, although prospective studies are warranted to assess the clinical utility of different molecular diagnostic methods.
Assuntos
Neoplasias Colorretais/diagnóstico , Melanoma/diagnóstico , Células Neoplásicas Circulantes , Neoplasias da Próstata/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Creatina Quinase/genética , Amplificação de Genes , Humanos , Masculino , Monofenol Mono-Oxigenase/genética , Antígeno Prostático Específico/genética , RNA Mensageiro/análise , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
We have applied the polymerase chain reaction to detect differences in relative amount of allele-specific transcripts of the low density lipoprotein receptor gene in individuals with heterozygous familial hypercholesterolemia (FH). This method is based on detection of loss of heterozygosity of exon-specific restriction fragment length polymorphisms present in amplified mRNA fragments as compared to amplified genomic DNA fragments. We detected loss of allele-specific mRNA in 20% of informative FH patients. In principle, this method enables the analysis of relative allele-specific transcript levels of any expressed gene and can thus be generally applied to study processes involving differential gene expression.
Assuntos
Alelos , Hiperlipoproteinemia Tipo II/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , RNA Mensageiro/genética , Receptores de LDL/genética , Transcrição Gênica , Sequência de Bases , DNA/sangue , DNA/genética , DNA/isolamento & purificação , Triagem de Portadores Genéticos , Humanos , Leucócitos/fisiologia , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , RNA/sangue , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/análise , Valores de Referência , Mapeamento por RestriçãoRESUMO
We have applied the denaturing gradient gel electrophoresis (DGGE) technique to detect sequence variations in exon 9 of the low density lipoprotein receptor (LDLR) gene in individuals with heterozygous familial hypercholesterolemia (FH). A fragment containing exon 9 and 25 base pairs (bp) of the intron boundary sequence at either side was amplified. To this fragment a 40-bp GC-clamp was attached by the polymerase chain reaction (PCR). We have analyzed a total of 165 DNA samples of FH patients and have detected a mutation in three cases. Two patients were found to have the previously described "South African" G to A transition in codon 408. In a third patient, we observed a different banding pattern of the DNA fragments on DGGE indicating a different mutation. The mutant homoduplex band of this sample was purified from the gel, cloned in an AT-vector and sequenced. Sequence analysis demonstrated a G to A transition of the consensus G-nucleotide at the intron 9 splice donor site. Cosegregation between this mutation and elevated plasma cholesterol levels was observed in family members of this FH patient. This mutation probably prevents normal splicing of the mRNA and represents the first identified splice-site mutation in the LDLR gene. We conclude that the use of DGGE of GC-clamped PCR-amplified exon sequences offers a general strategy for the detection of disease-producing mutations in the LDLR gene.
Assuntos
Hiperlipoproteinemia Tipo II/genética , Mutação Puntual , Receptores de LDL/genética , Sequência de Bases , Análise Mutacional de DNA/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Éxons , Humanos , Dados de Sequência Molecular , Desnaturação de Ácido Nucleico , Linhagem , Reação em Cadeia da Polimerase/métodos , Splicing de RNARESUMO
The present study investigates the relationship between mitochondrial activity and the expression of the BCL-2 gene in a panel of six human and murine leukemia/lymphoma cell lines. The cell lines all contained normal glucocorticoid receptors but differed widely in sensitivity to dexamethasone, ranging from very sensitive S49 lymphoma to completely resistant HL-60 acute leukemia cells. In this panel, 10- to 15-fold differences in basal adenosine triphosphate (ATP) content and adenosine diphosphate (ADP)/ATP ratio were correlated with up to fivefold differences in bcl-2 protein (in human cells) and approximately 25-fold difference in bcl-2 mRNA content (all cell lines). Moreover, ATP content and BCL-2 gene expression were inversely correlated with glucocorticoid sensitivity and cell cycle length. In resistant cell lines, sensitivity to dexamethasone was restored by the mitochondrial inhibitors rotenone and meta-iodobenzylguanidine. This sensitization was not accompanied by detectable reductions in bcl-2 mRNA or protein content, suggesting that the inhibitors were capable of overriding BCL-2-mediated inhibition of apoptosis. Increased mitochondrial activity and (overexpressed) BCL-2 appeared closely related properties of glucocorticoid-resistant cells, sharing common cellular targets in hormone-induced apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Dexametasona/farmacologia , Expressão Gênica , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proto-Oncogenes , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Western Blotting , Linhagem Celular , Humanos , Cinética , Leucemia Experimental , Leucemia Promielocítica Aguda , Linfoma , Camundongos , Proteínas Tirosina Quinases/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2 , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Receptores de Glucocorticoides/análise , Receptores de Glucocorticoides/metabolismo , Rotenona/farmacologia , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Tonsillar squamous cell carcinomas (a total of 14) were examined both for the presence of human papillomavirus (HPV) DNA and for p53 alterations. General primer-mediated HPV polymerase chain reaction (GP-PCR) revealed the presence of HPV DNA in 12/14 cases. Subsequent typing by HPV type-specific PCR and sequence or hybridization analysis of GP-PCR products revealed DNA from HPV 16 in seven cases, from HPV 33 in two cases, and from HPV 7, HPV 16/33 and HPV 33/59 each in a single case. p53 immunohistochemistry performed on nine HPV containing tonsillar carcinomas using polyclonal serum CM-1 showed elevated p53 levels in four cases. These included 3/5 HPV 16 containing carcinomas and the HPV 33/59 containing carcinoma. Analysis of p53 mutations using denaturing gradient gel electrophoresis (DGGE) of GC-clamped PCR products of exons 5 to 8 showed p53 gene alterations in 3/13 cases, including 2/11 HPV positive cases and 1/2 HPV negative cases. The alterations included a silent point mutation within exon 8 of an HPV 16 containing carcinoma, a 1 bp deletion within exon 8 of an HPV 33 containing carcinoma, and a missense mutation within exon 7 of one of the HPV negative carcinomas. There was evident discrepancy between p53 immunohistochemistry and gene analysis. Four HPV containing cases showing elevated p53 levels did not reveal the presence of exon 5 to 8 alterations affecting the amino acid code, suggesting the presence of mutations occurring in other exons or non-mutational p53 stabilization. The data indicate that HPV and elevated p53 can coexist in tonsillar carcinomas and that despite the low frequency of p53 mutations the presence of HPV is not exclusively related to the absence of mutated p53.