Sample Application for Tissue Microarrays

A. CONTACT INFORMATION:

B. SCIENTIFIC RATIONALE:

1. Introduction:

The importance of the RECEPTOR family in growth of breast cancer cells is supported by over twenty years of study of family members R1 and R2. The role of the R3 receptor has been less studied, although work from a number of laboratories has indicated that it is important for breast cancer cell proliferation. Our laboratory has been interested in LIGAND-induced signals that are transmitted by the R3 receptor. R3-LIGAND interactions lead to cellular proliferation, growth inhibition or differentiation by stimulation of a cascade of signaling events. To better understand LIGAND-mediated signal transduction pathways initiated by the R3 receptor, we performed a yeast two-hybrid screen using the non-phosphorylated cytoplasmic domain of R3 to identify R3-interacting proteins. We isolated a protein MARKER1 that interacts with the juxtamembrane domain of R3. MARKER1 is the human homologue of a previously identified cell cycle regulated mouse protein M-MARKER1. MARKER1 has several important structural motifs, including potential ... and ... phosphorylation sites, an amphipathic helical domain predicted to mediate protein/protein or protein/DNA interactions, and an ... motif present in many nuclear receptor binding proteins. Treatment of serum-starved breast cancer ... cells with LIGAND resulted in dissociation of MARKER1 from R3 and translocation from the cytoplasm into the nucleus . Ectopic expression of MARKER1 in R2, R3 expressing breast carcinoma cell lines resulted in inhibition of colony formation, a decreased proliferation rate, an accumulation of cells in the G2/M phase of the cell cycle, and suppression of growth in soft agar. In addition, a more differentiated phenotype was observed in ... breast cancer cells, as evidenced by increased expression of lipid droplets and of the milk protein casein. Thus, the overexpression of MARKER1 mimicked many of the differentiative effects of LIGAND in this system. MARKER1 represses transcription of cell cycle regulated genes that have ... consensus elements in their promoters. MARKER1 contains an autonomous C terminal transcriptional repression domain that binds ... and ....The ability of MARKER1 to repress transcription is partially reversed by ... inhibitors. MARKER1 binds to the ... promoter in a complex with ... transcription factor. Of interest, the ability of MARKER1 to repress transcription of cell cycle related genes was enhanced by LIGAND treatment, further supporting its role as an LIGAND regulated gene.

2. Specific Aim:

Although the ability of MARKER1 to induce differentiation of human breast cancer cells has been demonstrated in vitro, we have not studied MARKER1 expression in vivo in human breast cancer samples. However, work using a commercial tissue microarray indicates that MARKER1 is strongly expressed in normal human breast ductal epithelial cells. As MARKER1 expression in vitro is associated with a more benign phenotype, we hypothesize that MARKER1 may be decreased in breast cancer and that its expression may be associated with malignant progression. Interestingly, preliminary data on a limited number of samples suggest that MARKER1 expression is decreased in DCIS as compared to normal controls. We would like to perform a pilot project using the CBCTR tissue microarray to determine if there are any differences in expression of MARKER1 in normal versus tumor cells at different stages of malignant progression.

3. Marker to be tested:

C. TECHNICAL APPROACH:

1. Description of the Approach:

To test this hypothesis that MARKER1 protein expression may be altered in breast cancer, we will assess MARKER1 protein l evels and subcellular localization by immunohistochemistry using an affinity purified polyclonal antibody against MARKER1. This antibody has been extensively tested and detects only one band of the predicted molecular weight. The antibody is also commercially available through Company A. Both the localization and levels of expression will be assessed by Drs. John Doe and Mary Jones of the Pathology Department who have been actively engaged in this project. Our initial results indicate that antigen retrieval is not necessary for detection of MARKER1 protein. Endogenous peroxidase activity is blocked by immersion of tissue sections in a 3% solution of hydrogen peroxide in methanol at room temperature. Slides are incubated at room temperature with the MARKER1 antibody diluted 1:200 for one hour. All staining procedures are performed on a Ventana Benchmark automated slide processing equipment using the standard avidin-biotin method. Peroxidase activity is localized by the diaminobenzidine tetrachloride peroxidase reaction with Harris hematoxylin as a counterstain.

2. Evidence that specific techniques worked for paraffin embedded tissues:

We have tested a number of normal breast tissues from commericial arrays made from paraffin embedded tissue. We have provided an image of a human breast specimen from a Clinomics low density tissue array of normal tissue stained with MARKER1 antibody. We have also used MARKER1 antibody on commercial prostate arrays (Clinomics and Zymed) and have found good staining with no background. In addition, we have successfully stained paraffin embedded human tumor xenografts with the antibody.

3. Scoring System:

Immunohistochemical staining will be recorded by a semi quantitative and cells showing a positive reaction. Intensity will be recorded as 0 (no staining) to 3 (strong staining). The percentage of stained cells will be recorded as 0 (no tumor cells positive), 1 ( positive staining in 10% of tumor cells), 2 (positive staining in 10 –50% of cells) and 3 (positive staining in 50 % of tumor cells). To be scored as negative, a maximum of 10% of cells should be very weakly stained. Staining indexes will be calculated as the product of the staining intensity and proportion of cells stained.

4. Controls:

For a negative control, one slide will be stained with biotinylated secondary antibody and the avidin-peroxidase reagent, but no primary antibody. The positive controls will consist of the normal breast tissue on the slide.

D. TOTAL NUMBER OF SECTIONS:

I am requesting 4 replicate sections. One section will be used as a negative control. The other three will be from the 3 replicate blocks. Scoring will be done on all three blocks and averaged for individual tumor specimens. Our aim is to compare marker prevalence across stage groups as has been designed.