摘要:

Determination of cell lineage in acute leukemias is essential for diagnosis and treatment. Detection of myeloperoxidase (MPO) mRNA establishes myeloid lineage of leukemic blasts that may be too primitive to be identified as myeloblasts based on morphology, cytochemistry, or im-munophenotype. A highly specific and sensitive new procedure for MPO mRNA detection has been developed using HL-60 cells. It involves a microprocedure for total cellular RNA extraction, reverse transcription, and specific amplification of target sequences in the resulting MPO cDNA. by the polymerase chain reaction. Specific primers are designed to amplify an 89-base pair (bp) sequence from the signal peptide, 179 and 318-bp sequences from the start and end, respectively, of the heavy-chain sequence, and a 255-bp sequence overlapping the prore-gion and light chain. The correct-size amplification products. detected electrophoretically, demonstrate MPO mRNA expression in the leukemic cells analyzed. The sensitivity of this new procedure was evaluated on serial concentrations of HL-60 cells and was found to be 10–104cells depending on the MPO cDNA amplified sequence. No amplification products were obtained using peripheral blood lymphocytes as a negative cellular control. The specificity of the procedure is demonstrated by Southern blotting and hybridization with32P-labeled oligonucleotide probes specific for each of the amplified sequences. An additional advantage of this procedure is availability of results in 8–24 h, compared with 1–2 weeks for conventional RNA methods.

ISSN:1052-9551    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

The matrix metalloproteinase enzymes have been implicated in tumor invasion and metastasis by a series of correlative immunohistochemical studies. In addition, direct evidence for the role of these enzymes in this pathologic process comes from studies using specific metalloproteinase inhibitors to block tumor invasion and metastasis formation, both in vitro and in vivo. Synthetic oligonucleotide primers for four metalloproteinases (MMP-1. MMP-2, MMP-9. MMP-10) and their tissue inhibitors (TIMP-1. TIMP-2) were selected, synthesized, and optimized in the reverse transcriptase-polymerase chain reaction (RT-PCR) to study the qualitative profile of these enzymes and inhibitors in cultured human tumor cells and tumor tissues. These primers are specific and generate unique amplification products for each appropriate enzyme and inhibitor. Slight enhancement in the amplification of cDN A products was achieved by adding di-methylsulfoxide to the reaction mixture, but commercial enhancement reagents were ineffective. Using this RT-PCR method, cDNA amplification was successful with RNA from as few as 20 cultured tumor cells. The RT-PCR analysis was done on three invasive human colon adenocarcinomas and their paired adjacent normal mucosa. The results show MMP-1 and MMP-2 products in all three tumors, and MMP-2 detected in one of the three normal mucosa samples; TIMP-2 expression was present in two of three patients and awaits quantitative assessment of RT-PCR products.

ISSN:1052-9551    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

Several studies have implicated the extracellular matrix-degrading metalloproteinases (MMPs) as essential agents in tumor cell invasion and metastasis. In the present study, we have investigated the patterns of expression of a number of MMPs and their specific tissue inhibitors (TIMP-1 and TIMP-2) in human colonic tissue samples that represent various stages of progression from adenomas showing different degrees of dysplasia to adenocarcinomas. We assessed levels of mRNA by Northern blot analysis and the results were measured semiquantitatively by densitometry. In total, we analyzed nine adenomas of varying size and with varying degrees of dysplasia. three adenomas with adenocarcinoma (malignant polyps). and five adenocarcinomas. Although expression of MMP and TIMP mRNA was highly intercorrelated, transcripts for stromelysin 3 and TIMP-2 (high) showed the strongest relation to the neoplastic process. Detection of stromelysin 3 mRNA accompanied a diagnosis of severe dysplasia or malignancy, whereas levels of TIMP-2 (high) mRNA transcripts permitted finer distinctions on the neoplastic continuum. These data indicate changes within extracellular matrix acquired during the process of malignant transformation of human sporadic colorectal neoplasia.

ISSN:1052-9551    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

We determined loss of heterozygosity from formalin-fixed, paraffin-embedded tissue of colorectal carcinoma using microsatellite polymorphism. The polymorphism was assayed based on DNA amplification by the polymerase chain reaction (PCR). The PCR-analyzed micro-satellite method was applied to assay degraded DNA extracted from paraffin-embedded blocks with adenocarcinoma of colon. The DNA from 26 tumors as well as their corresponding normal tissue samples were successfully amplified using a dinucleotide microsatellite located within an intron of the deleted in colorectal carcinoma gene. Allele losses on this marker were detected in 33% of informative colorectal carcinomas. This study demonstrates that microsatellites provide a powerful set of DNA markers for loss of heterozygosity on archival specimens.

ISSN:1052-9551    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

Trisomy of chromosome 12 has been frequently described in various neoplasms, particularly in tumors of the female genitourinary tract. Fluorescence in situ hybridization with a centromeric repetitive DNA probe, specific for chromosome 12, was done to detect such cytogenic changes on frozen-tissue sections from 10 cases of ovarian sex cord stromal tumors. The case series was composed by granulosa cell tumors (four cases), fibromas (four cases), thecoma (one case), and Sertoli-Leydig cell tumor (one case). In granulosa cell tumors, the range of trisomy was 12 to 3297 and in fibromas 8 to 229r, whereas in the single case of thecoma trisomy was present in 8rr and in the Sertoli-Leydig cell tumor in 4ryc of the nuclei examined. These results represent an additional series of cases of trisomy 12 in ovarian neoplasms, namely, in ovarian sex cord stromal tumors.

ISSN:1052-9551    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

As part of a retrospective study into the prevalence of the to 14.18) translocation in B-cell lymphomas, we assessed the suitability of the polymerase chain reaction (PCR) to amplify the U 14.18) major breakpoint region (MBR) in frozen and formalin-fixed tissue. Considering Southern blotting as a standard, the sensitivity of PCR was 81%. Of the various procedures used to extract DNA from paraffin-embedded tissue (PET), proteinase K digestion in the presence of nonionic detergents gave the highest yield and quality of DNA and the most efficient amplification rate. Using this method, excellent amplification rates (100%) were obtained for both the beta-globin control sequence and the MBR t(14;18) for fixed follicular lymphoma specimens collected in the previous 2 to 6 years (n = 27). Of nine older PETs. PCR on six gave inconsistent results, probably because of the poorer-quality substrate used for amplification. Specimens exposed to for-mol sublimate or formalin-acetic acid-alcohol were as suitable for amplification as tissues fixed in neutral-buffered formalin. The overall incidence of the MBR t(14.18) in all follicular lymphoma specimens as detected by both Southern blotting and PCR was 59%; (23 of 39).

ISSN:1052-9551    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

A highly sensitive in situ hybridization procedure was established using digoxigenin-I I-dUTP-labeled probes that were prepared by the polymerase chain reaction (PCR). By using 12 sets of primers,BamHI-Wfragment of the Epstein-Barr Virus (EBV) was amplified with labeled substrate in individual PCRs. Then the 12 probes (average size. 120 base pairs) were mixed and hybridized with cultured and RNase-treated Namalwa cells, which contain two copies of EBV genomes per cell. The strength of the signals was much stronger as compared with random-primed probe. Our results indicate that the size-averaged PCR probes magnified the sensitivity for detecting low copy numbers of virus genomes by in situ hybridization and that this technique has the potential for investigating latent virus infection in other clinical situations.

ISSN:1052-9551    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

There have been conflicting reports regarding the association of cytomegalovirus (CMV) and recurrent spontaneous abortions. It is difficult to assess the role of CMV in the endometrium by histology alone, since the characteristic cytomegalic virocytes are often scarce or absent in this site. Our purpose was to use the polymerase chain reaction (PCR) to detect cytomegalovirus in gestational tissue of women with recurrent spontaneous abortions. DNA was extracted from 25 samples of paraffin-embedded, formalin-fixed gestational tissue from 21 women with at least three unexplained spontaneous abortions (mean, 3.4). DNA from an unstained paraffin section of each specimen was amplified using nested, multiplex PCR specific for the late antigen and the major immediate early genes of CMV The assay used has a demonstrated level of sensitivity on the order of 102virocytes per square centimeter of 4-p.M paraffin section. Intact DNA was successfully isolated from 21 specimens in 18 patients. Histologic features of CMV infection were completely absent from these cases, and none of these specimens contained evidence of cytomegalovirus DNA. These findings suggest that CMV infection of gestational tissue is not a common direct cause of recurrent spontaneous abortions.

ISSN:1052-9551    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

The reverse transcriptase polymerase chain reaction (PCR) is a technique for the study of gene expression that requires far less RNA for analysis than Northern blots. The inclusion of cRNA standards in the initial reverse transcription step is a way to control for the tube-to-tube variation often inherent in the technique and to permit quantitation of the starting amount of the native mRNA being analyzed. We describe a method using overlapping oligonucleotides to produce templates for the production of cRNA standards for up to three different mRNA species. The first step is the synthesis of a pair of overlapping oligonucleotides each of which encodes, respectively, sequences analogous to either sense or antisense primers for the PCR amplification of up to three different messages. These oligonucleotides are designed to have complementary 3' ends which permit spontaneous annealing and allow subsequent mutually priming extension of the annealed double-stranded portion by T7 DNA polymerase. The T7 and SP6 RNA polymerase promoters are then added to the ends of the template using standard PCR techniques. Once the template is assembled, T7 and SP6 RNA polymerases are used to produce copious quantities of cRNA standards and controls. This technique can be used to construct multiple cRN A standards for essentially any messages of interest. Production of cRNA by a single T7 RNA polymerase reaction yields standards sufficient for several thousand separate reverse transcriptions.

ISSN:1052-9551    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

Oligonucleotides used in in situ hybridization (ISH). regardless of their sequence specificity, bind to neuroendocrine (NE) cells in normal gastrointestinal mucosa and tumors. This nonspecific binding, presumably related to the presence in NE cells of hidden NH2:groups of obscure origin, can be prevented by acetic anhydride treatment of the sections. This is a routine step in several ISH protocols but not in all. This study emphasizes the need to establish safe protocols and controls to check the specificity of ISH procedures.

ISSN:1052-9551    

出版商:OVID    

年代:1993

数据来源: OVID