Biomedical subjects

M A Lane

Publications and source records attributed to M A Lane.

114 records · Page 7Linked to original sources

PubMedDate not supplied

Inhibitors of the polymerase chain reaction in Papanicolaou stain. Removal with a simple destaining procedure.

OBJECTIVE: To identify inhibitors of the polymerase chain reaction (PCR) within Papanicolaou stain and to develop a method of removal. STUDY DESIGN: PCR was performed on cellular digests of archival, Papanicolaou-stained cervical smears and unstained cervical smears using primers to the human beta-globin gene to determine if Papanicolaou stain inhibited the reaction. Next, in order to identify the specific reagents responsible for inhibition, components of the Papanicolaou stain were added to PCRs performed on purified human genomic DNA. Finally, PCR was performed on cellular digests of cervical smears that had been destained using 1% HCl to determine if the inhibitors of PCR were removed. RESULTS: Cellular digests from Papanicolaou-stained cervical smears did not yield products from PCR, whereas cellular digests from unstained cervical smears always yielded PCR products. Analysis of individual Papanicolaou stain reagents revealed inhibition of PCR by hematoxylin and aluminum sulfate. These inhibitors could be removed from Papanicolaou-stained cervical smears by destaining the slides with 1% HCl. CONCLUSION: The hematoxylin and aluminum sulfate within Papanicolaou stain inhibit PCR but can be removed from Papanicolaou-stained cytologic specimens using a simple destaining procedure.

Alum Compounds↗

PubMedDate not supplied

Immunohistochemical analysis of BRCA1 expression in normal human buccal cells.

While tumor suppressor properties of BRCA1 are well documented, less is understood concerning the normal function of this protein in healthy adult tissues. We demonstrate here that BRCA1 protein is expressed in human buccal cells. Expression of BRCA1 mRNA transcripts in buccal cells was confirmed by PCR of reverse transcribed RNA. Three BRCA1 antibodies, D20, MS110 and MS13, reacted positively with cytospin deposited buccal cells, demonstrating apparent cytoplasmic and nuclear distribution of BRCA1 protein. Double antibody immunofluorescent, binding of D20 and MS110 was differentially distributed. We conclude that buccal cells provide a cell source for exploration of BRCA1 protein function in normal human adults.

Adult↗

PubMedDate not supplied

Spontaneous conversion to estrogen receptor expression by the human breast epithelial cell line, MCF-10A.

The human breast epithelial cell line, MCF-10A, derived from tissue from a woman undergoing a cutaneous mastectomy for fibrocystic breast disease, is negative for estrogen receptor expression, has undergone minimal genetic changes, retains many of the characteristics of normal breast epithelium and fails to exhibit growth in nude mice. When transfected with a functional copy of the estrogen receptor, both ER and MDM2 expression are negatively regulated by the presence of increasing concentrations of estradiol, as previously reported. We obtained the MCF-10A cell line from the American Type Culture Collection and confirmed that it was negative for ER expression. After approximately 20 passages under differing growth conditions, one subline was determined to be positive for ER expression. Growth of this ER-positive subline in phenol red-free media supplemented with charcoal-dextran stripped serum in the presence of nanomolar concentrations of estradiol failed to modulate ER and MDM2 expression, and induced expression of both pS2 and cathepsin D. Simultaneously with these observations, we observed that this subline, unlike the parent MCF-10A line, overexpressed P53 protein with a nuclear localization. Intermediate levels of the P53-inducible protein p21 WAF1/Cip1 were also detected in the ER-positive subline whereas levels of this protein in the parent subline were barely detectable, as measured by immunohistochemical methods. We conclude from these studies that ER expression and P53 alteration may constitute early steps in progression of malignant potential for breast cancer development.

Breast Neoplasms↗

PubMedDate not supplied

An antibody assay predictive of BRCA1 mutations in ovarian tumors and normal tissue.

To determine whether immunohistochemistry can identify BRCA1 mutations, immunohistochemical (IH) analysis was undertaken on paraffin sections of paired ovarian cancer and normal tissue using antibodies against both terminal regions of the BRCA1 protein. Ten patients at risk for BRCA1 mutations were studied. The results of BRCA1 mutation analysis and IH were compared. In tumor, IH correctly identified the presence or absence of loss of heterozygosity in all specimens. In all uninvolved specimens, IH correctly identified the presence or absence of a germline mutation. The sensitivity, specificity, positive and negative predictive values were 100% suggesting promise as a rapid and inexpensive screen.

Adult↗

PubMedDate not supplied

Immunohistochemical analysis of BRCA2 expression in normal human buccal cells.

Previously we demonstrated that protein coded by the BRCA1 gene was expressed in normal human buccal cells. The present study confirmed that BRCA2 protein was similarly expressed in these cells. Messenger RNA for BRCA2 was detected with sequential use of two primer sets. Pooled cell samples from healthy donors reacted strongly with two commercially available antibodies, I17 and C15. Immunoreactivity was present in both cytoplasmic and nuclear compartments. We conclude buccal cells will provide a suitable model for exploration of normal BRCA function.

Adult↗

PubMedDate not supplied

A rapid immunoassay predicts BRCA1 and BRCA2 mutations in buccal cells.

We developed a rapid, non-invasive, and inexpensive, assay capable of identifying BRCA1 and BRCA2 (BRCA) mutations in buccal cells. To determine the predictive value of this immunoassay, a double blind study of 13 high risk individuals was conducted by two independent teams. As greater than 90% of BRCA mutations result in protein truncations, a diminished anti-carboxy immunoreactivity relative to anti-amino immunoreactivity was scored as predictive for mutation. Comparison to BRCA DNA analysis was undertaken. The positive and negative predictive values were 90% and 100% respectively (p<0.02), suggesting great promise as an inexpensive and rapid screen for BRCA mutations.

BRCA1 Protein↗