ISSN:1052-9551    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

The polymerase chain reaction (PCR) is a rapid and highly sensitive method for detection of a variety of chromosomal translocations in malignant tissues. Detection of each different type of translocation, or even DNA rearrangements at different breakpoint cluster regions within the same type of translocation, usually requires separate thermocycling parameters and/or buffer conditions. In this report, we describe a single set of reaction conditions, making use of progressively decreasing annealing temperatures and a standardized reaction buffer, that permits the detection of several different translocations simultaneously. Specificity equal to or better than current procedures and sensitivity equivalent to one malignant cell in 1 |MX 105normal cells was achieved for translocations t(14;18)(q32; q21), t(9; 22)(q34: q 11), and t(4; l1)(q21; q23). For PCRs formerly requiring different, fixed annealing temperatures, the new technology allows batching or multiplexing of PCR samples. Thus, shorter turnaround time, decreased cost per sample, and simplified mechanization of PCR may be attainable using this assay.

ISSN:1052-9551    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Trk receptors have been identified by immunohisto-chemical methods in primitive neuroectodermal tumor (PNET)/Ewing's sarcoma (ES). However, the presence of different members of the Trk family of receptors in PNET/ES has not been specified. We have examined whether Trk A, B, and C receptors are specifically expressed in ES both with and without features of neural differentiation. Ten ES tumors (five primary tumors of bone and five extraosseous tumors transplanted into nude mice) were investigated for expression of Trk receptors by immunohistochemistry and reverse transcription-polymerase chain reaction. One primary ES and the five grafted ES tumors exhibited signs of neural differentiation; the remaining four primaries were undifferentiated ES. Other tumor types were analyzed as controls; they included three neuroblastomas (NB), two lymphomas, and single cases of pheochromocytoma (PHEO), schwannoma (SCHW), osteosarcoma, and carcinoma of breast, colon, and kidney. Trk receptors were detected in paraffin-embedded tumor tissue sections by means of a pan-Trk polyclonal antibody raised against the 14 carboxy-terminal residues of gpl40trk, andtrkA, B, and C transcripts were specifically detected by polymerase chain reaction-based amplification on cDNAs derived from tumor RNA with MuLV reverse transcriptase. Reactivity to the pan-Trk antibody was exhibited by six ES tumors, the three NBs, and the single PHEO and SCHW cases; immunoreactivity was restricted to differentiated tumors, in the case of ES. Tumor types positive for immunostaining were also distinguished by containing transcripts of TRK genes. However, thetrkA, B, and C expression pattern of ES differed from that of NBs, PHEO, and SCHW. Transcripts oftrkA, B, and C were detected in seven, four, and one case of ES, respectively, and in five, two, and five cases of NB, PHEO, and SCHW, respectively. Interestingly, all differentiated ES tumors containedtrkA transcripts. Tumors of neuroectodermal phenotype and/or derivation were thus characterized by a distinct consensus expression pattern:trkA+/B-/C-for differentiated ES andtrkA+/B-/C+for NB-PHEO-SCHW. These results indicate that theTRKgene family is frequently activated in ES; they also suggest that Trk A receptor is a feature of ES with neural differentiation, whereas Trk B and C receptors seem to be present in undifferentiated ES.

ISSN:1052-9551    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

H19 is an imprinted maternally expressed gene, which is not translated to protein and functions as an RNA molecule. It is closely related to the oppositely imprinted paternally expressed insulin-like growth factor 2 (IGF-2). While the biological function of H19 is not understood, IGF-2 is a growth factor that plays a role in human follicular and endometrial differentiation. We examined the expression of H19 in the endometrium and ovary during the menstrual cycle by in situ hybridization applied to paraffin sections of human endometrium and ovaries at different stages of differentiation. In the endometrium, H19 expression was confined to the stroma and fluctuated with endometrial dating to reach its peak in the late secretory stage. IGF-2 was also prominently expressed in late secretory endometrium, but its expression was evident both in the stroma and glandular epithelium. Expression of H19 was not found in primordial, primary, and preantral follicles of the ovary, but prominent expression was evident in the theca of antral and cystic atretic follicles, and focal expression was noted in the granulosa of corpora lutea. An association between H19 expression during the menstrual cycle and the differentiation state of the human female reproductive tract, which is under hormonal control, is suggested.

ISSN:1052-9551    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

To evaluate the specificity and applicability to the study of human tumor cells of the reverse transcription (RT) in situ PCR and RT polymerase chain reaction (PCR) in situ hybridization techniques, we examined five melanoma cell lines and five nonmelanoma lines for tyrosinase mRNA using primers specific for tyrosinase. Each procedural step was optimized and minutely controlled, and results from the in situ techniques and solution-phase RT-PCR were compared. All melanoma lines showed a specific pattern of perinuclear cytoplasmic reaction not seen in nonmelanoma lines. There was exact agreement between the results from the RT in situ PCR and RT-PCR in situ hybridization techniques and those from solution-phase RT-PCR. Ribonuclease digestion abolished cytoplasmic staining, as did omission of the reverse transcriptase step. Nuclear staining was seen in melanoma and nonmelanoma lines, apparently as a result of DNA synthesis from repair-replication and mispriming or nonspecific amplification. Neither high concentrations of deoxyribonuclease nor long incubation periods abolished this effect completely. Demonstration of cytoplasmic mRNA by RT in situ PCR and RT-PCR in situ hybridization specifically identifies cells of melanocytic lineage.

ISSN:1052-9551    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Current research into cytokine production in tissue sections relies on the detection of cytokine proteins using a variety of immunohistochemical methods. The disadvantages of this technique are that precise localization to a particular cell is difficult and it is uncertain whether the cells detected by this method are the origin or target of the cytokine or rather have nonspecifically absorbed the secreted cytokine. This question can be clarified using in situ hybridization, but current techniques are insensitive, poorly localizing, or time consuming. Biotin-labeled riboprobes were generated from cDNA fragments sandwiched between two RNA polymerase promoters (SP6 and T7 RNA polymerases) using a commercial riboprobe generation kit containing biotin-labeled UTP. The in situ hybridization technique was used to demonstrate cytokine mRNA in a range of tissues containing an inflammatory infiltrate and with a range of cytokine probes. This technique of in situ hybridization was combined with immunohistochemistry using an immunoalkaline phosphatase technique to show the powerful combination of these two techniques. The biotin-labeled riboprobes were sensitive enough to detect a range of cytokine mRNAs in a variety of tissue sections. The technique can be completed over a 24-h period and produces a stable color product that can be stored for long periods and can be quantitated using image analysis techniques. This technique was performed on paraffin-embedded tissue as well as cryosections and allowed for the detection of mRNA in archival tissue. It was also successfully combined with immunohistochemical techniques to determine simultaneously the localization of a cytokine product in particular cell lineages. A nonradioactive method for in situ hybridization using biotin-labeled riboprobes is described; it is capable of detecting mRNA products from a range of genes in a variety of tissue samples. An amplification step in the method enhances the sensitivity to a level that approaches that of radioactive methods, while maintaining the speed, safety, and simplicity of an immunoperoxidase detection system. The ability to use paraffin-embedded tissue with this method allows for improved tissue architecture and examination of archival tissue. These features should ensure greater use of in situ hybridization techniques in future research studies.

ISSN:1052-9551    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Retinoids modulate gene activity, cell growth, and differentiation by binding to a series of nuclear receptors, i.e., retinoic acid receptors (RARs) or retinoid X receptors. Retinoic acid (RA) inhibition of estrogen receptor (ER)-positive breast carcinoma seems to be mediated through RARα. Estrogens upregulate RARα in ER-positive breast carcinoma cell lines. In this study we examined RARα expression in the ER-positive MCF7 and ER-negative MDA-MB-231 human breast carcinoma cell lines as well as in 10 ER-negative and 9 ER-positive infiltrating ductal breast carcinoma specimens using immunohistochemistry and quantitation by image cytometry. MCF7 cells expressed twofold higher levels of RARα protein than MDA-MB-231 cells. RARα expression, as detected by immunostaining and quantitated by image cytometry, was upregulated in these cells by estradiol. ER-positive breast carcinoma specimens also exhibited approximately twofold higher RARα levels than their ER-negative counterparts. Thus, RARα expression is significantly elevated in ER-positive breast tumors as assessed by detection and quantitation using immunohisotchemical staining and image cytometry, respectively. Whether the decrease in RARα protein levels and loss of RA-mediated growth inhibition in ER-negative tumor plays a role in the increased metastatic potential of ER-negative tumors remains to be determined.

ISSN:1052-9551    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

PCR assays for the presence of mutant K-rasor p53 sequences are potentially useful as sensitive tests for tumor diagnosis. The technical challenge is to design assays sensitive enough to detect a few molecules of mutant DNA yet sufficiently specific that a false positive signal is not produced by a 105- or 106-fold excess of normal DNA. We determined the detection limit of allele-specific PCR (ASA) as a function of the particular mismatch involved using all 12 possible mismatches in two different DNA sequence contexts (K-rascodon 12 and p53 codon 273). Depending on the identity of the mismatch, mismatched template was amplified 102-104-fold less than perfectly matched template. In other words, a mutant allele could be detected by ASA if it represented > 1–0.01 of the total DNA from that locus. Peptide nucleic acid (PNA) clamping was used to improve the K-rasASA assay. Selective amplification of mutant sequences was achieved using a PNA complementary to the normal sequence to inhibit the amplification of wild-type DNA. PNA clamping followed by ASA resulted in significant improvement in sensitivity and specificity, permitting the detection of tumor DNA diluted with a 300,000-fold excess of normal human DNA.

ISSN:1052-9551    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

Specimens of a vacuum curettage were microscopically indicated for a hydatidiform mole. The combination of three different approaches identified the specimen as a partial mole caused by the fertilization of a haploid ovum by sperm containing a haploid or diploid nucleus with one or two sets of paternal genetic material. Interphase fluorescence in situ hybridization identified three chromosome 1 centromeres, and DNA flow cytometry revealed a peak with a DNA index of 1.50. The combination of flow cytometric cell sorting and microsatellite marker polymerase chain reaction proved that in this case two alleles were from paternal origin. Because it is known that partial hydatidiform moles have a tendency for recurrence, specimens from the same patient of an earlier executed vacuum curettage were investigated. Microdissection of the villi was performed before DNA isolation in this case, as too few villi were present for DNA flow cytometry and cell sorting. In this case, no evidence was fond for additional alleles. This study shows the diagnostic potential of microsat-ellite markers for genetic typing of hydatidiform moles.

ISSN:1052-9551    

出版商:OVID    

年代:1997

数据来源: OVID

摘要:

We compared PCR-SSCP detected mutations of k-ras (codon 12) and p53 (exons 5–8) to ERBB-2 immunostaining and clinicopathologic features in 31 pulmonary adenocarcinomas. There were nine tumors (29) with mutations of ras, 13 tumors (42) with mutations of p53, and three tumors (10) with mutations of both. Neither k-ras nor p53 mutation alone was significantly correlated with stage, grade, or survival. However, tumors with k-ras mutation were more frequently associated with an invasive growth pattern, defined as >30 tumor volume composed of infiltrative nests of cells within desmoplastic, scar-like stroma [

ISSN:1052-9551    

出版商:OVID    

年代:1997

数据来源: OVID