RESUMO
OBJECTIVE: To study the feasibility of utilizing mRNA recovered from cytologic Papanicolaou (Pap) specimens as a resource for gene expression studies of normal and diseased cells. STUDY DESIGN: To assess the effects of fixation on mRNA recovery and analysis, fresh Pap samples were processed by three separate methods: (1) routine cytologic fixation (2) 70% ethanol fixation, and (3) air drying without fixation. One-week-old, 1-month-old, 1-year-old and 10-year-old samples were studied to determine the quality of mRNA in archival samples. mRNA quality was analyzed by RT-PCR for the HPRT gene, and by complete transcript amplification. Both heterogeneous (whole slide scrapes) and microdissected cell populations were studied. RESULTS: Reverse transcriptase-polymerase chain reaction (RT-PCR) for the hypoxanthine guanine phosphoribosil transferase gene was positive in all fresh and archival samples and was not affected by fixative, processing methodology or microdissection. Complete transcript amplification followed by gel electrophoresis showed cDNA smears in all fresh samples with a maximum intensity between 1 and 2 kilobases (kb). Amplification of mRNA was not affected by fixation. Smaller cDNA smears were seen in archival specimens with a maximum intensity between 0.5 and 1.5 kb in both one-week-old and one-month-old samples. Smears of approximately 500 base pairs were observed in the 1-year-old and 10-year-old samples. Successful mRNA amplification was possible from microdissected cell populations. CONCLUSION: Messenger RNA recovery and analysis is possible from archival cytologic specimens, suggesting that they can serve as a useful template for RT-PCR analysis of individual genes as well as newly developing high-throughput gene expression methodologies, such as microarrays. Cytologic samples may be particularly useful for study of archival samples as well as diseases from which tissue samples amenable to mRNA-based studies are not available.
Assuntos
Colo do Útero/citologia , Colo do Útero/patologia , Teste de Papanicolaou , RNA Mensageiro/análise , Esfregaço Vaginal , Colo do Útero/enzimologia , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Estudos de Viabilidade , Feminino , Humanos , Hipoxantina Fosforribosiltransferase/genética , Inflamação , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
Cathepsins D, B, and L are acidic lysosomal proteinases involved in intracellular protein turnover. Increased levels of these enzymes have been reported to be indicators of aggressive tumor behavior in human and rodent tumors. In breast cancer increased levels of cathepsin D have been reported to be an independent prognostic factor in women with stage I disease. We used standard immunohistochemical techniques on formalin-fixed, paraffin-embedded tissue to examine the levels of cathepsins D, B, and L in 80 carcinomas of the breast and compared that with other indicators of aggressive tumor behavior, including stage of disease, tumor size, nuclear grade, estrogen receptor status, disease recurrence, and 5-year survival rates. Positive granular cytoplasmic staining was detected for cathepsin D in 90% of the tumors, for cathepsin B in two thirds of the tumors, and for cathepsin L in approximately one half of the tumors. Positive staining also was seen in normal breast epithelium, areas of apocrine metaplasia, stromal fibroblasts, and macrophages. Our results did not show a correlation between the expression of cathepsins D, B, and L and other indicators of aggressive tumor behavior. We conclude that the results obtained using polyclonal anticathepsin antibodies do not support the prognostic usefulness of immunohistochemical analysis of these three proteinases in tumor cells in human breast cancer.