DETECTION OF BREAST CANCER MICROMETASTASES IN BONE MARROW USING REVERSE-TRANSCRIPTASE CHAINREACTION AND SOUTHERN HYBRIDIZATION

Objective: The aim of this study was to detect micrometastases in bone marrow of primary breast cancer patients, and compare with other clinical parameters. Methods: Cytokeratin 19 (CK-19) gene mRNA expression was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot hybridization. Human breast cancer cell line T47D was mixed with bone marrow cells in different proportions. The positive detection rate was compared among RT-PCR, Southern blotting and immunohistochemistry (IHC) methods. Results: Cytokeratin 19 gene was expressed in all 6 positive control samples while the expression was not seen in 8 negative control samples. In all 54 patients 14 cases were CK-19 positive (25.9%) by RT-PCR, another positive signal was obtained in 5/54 (9.3%) of bone marrow samples by Southern blotting. The total positive cases are 19/54 (35.2%). CK-19 IHC+ cells were detected at a dilution of one T47D cell in 5×104 bone marrow cells, while the sensitivity detected by PCR and Southern blot hybridization was at 1∶5×105 and 1∶1×106, respectively. This demonstrates that RT-PCR and Southern blotting was at least 20 times more sensitive than the IHC method. The micrometastases positive rate of the larger tumor size group (>5.0 cm) was significantly (P<0.05) greater than that of the smaller tumor size group (0–2.0 cm). Conclusion: detection of micrometastases in bone marrow by RT-PCR and Southern blotting, using CK-19 as a biological marker, is highly sensitive and it is a method to be used for anticipating the prognosis of breast cancer patients.

COMBINED DETECTION OF BREAST CANCER MICROMETASTASES IN THE LYMPH NODES AND BONE MARROW USING REVERSETRANSCRIPTASE CHAIN REACTION AND SOUTHERN HYBRIDIZATION

Objective: The presence of lymph nodes and bone marrow micrometastases of patients with breast carcinoma by immunohistochemistry (IHC) methods has been strongly correlated to early recurrence and shorter overall survival. The aim of this study was to detect micrometastases in matched sample pairs of lymph nodes and the bone marrow of primary breast cancer patients using a more sensitive method, and compare with other clinical parameters. Methods: Cytokeratin 19 (CK-19) gene mRNA expression was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot hybridization. Human breast cancer cell line T47D was mixed with bone marrow cells at different proportions. The positive detection rate was compared among RT-PCR, Southern blotting and IHC methods. Results: Cytokeratin 19 gene was expressed in all 6 positive control samples, while the expression wasn’t seen in 18 negative control samples. CK-19 IHC positive cells were detected at a dilution of one T47D cell in 5×105 bone marrow cells, while the sensitivity detected by PCR and Southern blot hybridization was at 1:5×104 and 1:106, respectively. In the samples from the 35 patients, we found CK-19 positive cells in 2 cases (5.7%) by IHC. CK-19 gene expression signal was detected in 14/35 (40%) by RT-PCR, and 17/35 (48.6%) by southern blotting. Four cases were micrometastases positive both in lymph node and bone marrow (11.4%). There was no correlation between CK-19 detection and other clinical parameters. Conclusion: combined detection of micrometastases in lymph node and bone marrow by RT-PCR and Southern blotting, using CK-19 as a biological marker, is a highly sensitive method for breast cancer.

Preliminary Report of Molecular Detection of Retinoblastoma Gene Mutations

To develop gene diagnosis for retinoblastoma predisposition, it is necessary to disclose the retinoblastoma gene mutations or deletions in detail. Genomic DNA from tumor and peripheral white blood cells in 33 patients with retinoblastoma was detected with 3.8kb probe derived from 3' end of retinoblastoma gene cDNA. The gene abnormalities, including deletion, partial deletion and rearrangement, were found in 18 patients. Further research will be aimed at microdeletions or mutations for those patients wti...

Characterization of Episomal Replication of Bovine Papillomavirus Type 1 DNA in Long-Term Virion-Infected Saccharomyces Cerevisiae Culture

We have previously reported that bovine papillomavirus type 1(BPV-1) DNA can replicate its genome and produce infectious virus-like particles in short term virion-infected S. cerevisiae(budding yeast) cultures(Zhao and Frazer 2002,Journal of Virology, 76:3359–64 and 76:12265–73). Here, we report the episomal replications of BPV-1 DNA in long term virion-infected S. cerevisiae culture up to 108 days. Episomal replications of the BPV-1 DNA could be divided into three patterns at three stages, early active replication(day 3–16), middle weak replication(day 23–34/45) and late stable replication(day 45–82). Two-dimensional gel electrophoresis analysis and Southern blot hybridization have revealed further that multiple replication intermediates of BPV-1 DNA including linear form, stranded DNA, monomers and higher oligomers were detected in the virion-infected yeast cells over the time course. Higher oligomers shown as covalently closed circular DNAs(cccDNAs) are the most important replication intermediates that serve as the main nuclear transcription template for producing all viral RNAs in the viral life cycle. In this study, the cccDNAs were generated at the early active replication stage with the highest frequencies and then at late stable replication, but they appeared to be suppressed at the middle weak replication. Our data provided a novel insight that BPV-1 genomic DNA could replicate episomally for the long period and produce the key replication intermediates cccDNAs in S. cerevisiae system.