CL1 - Stories: Building on and Integrating Previous Knowledge



 
 
   
   
 
   
   
 
 
 
 
 
   
 
   
   


 
 
 
   
 
 
   
 
 
 
 


Building on and Integrating Previous Knowledge
 
- by Ann Burgess


Example of a Study Guide
  - prepared by Lynn Allen-Hoffmann


Breast Cancer: Gene-Cell-Tissue Interactions

Week 13, April 13-17, 1998

Investigating the Cellular Functions of Human Breast Cancer Gene 1 (BRCA1): Controversial Beginnings.

Research Papers:
Chen, Y. Chen, C.-F., Riley, D.J., Allred, D.C., Chen, P.-L., Von Hoff, D., Osborne, C.K., Lee, W.H. (1995) Aberrant subcellular Localization of BRCA1 in Breast Cancer. Science 270:789-791.

Scully, R., Ganesan, S., Brown, M., DeCaprio, J.A., Cannistra, S.A., Feunteun, J., Schnitt, S. Livingston, D.M. (1996) Technical Comment: Location of BRCA1 in Human Breast and Ovarian Cancer Cells. Science 272:123-125.

Chen, Y., Chen, P.-L., Riley, D.J., Lee, W.H., Allred, D.C., Osborne, C.K. (1996) Response: Location of BRCA1 in Human Breast and Ovarian Cancer Cells. Science 272:125-126.


Also Required
Weinberg, R.A. (1996) How Cancer Arises. Scientific American pp. 62-70.

Bishop, J.M. (1995) Cancer: The rise of the genetic paradigm. Genes and Development 9:1309-1315.


Learning Objectives
By the end of the week students should be able to:

    Explain the common mechanism by which diverse agents (X-rays, tumor viruses, carcinogenic chemicals, etc.) can lead to a common result: unregulated growth.

    BRCA1 may encode a transcription factor. Describe: (1) the structural features of BRCA1 that led the authors to hypothesize that it is a DNA-binding protein, and (2) how a mutant gene for a transcription factor might cause the uncontrolled growth of breast epithelial cells.

    Describe how the authors characterized the cellular location of BRCA1 protein and explain why this is important.

    Explain the difference between monoclonal antibody and polyclonal antisera and tell how each is prepared.

    Compare and contrast the Chen group's findings with the Scully group's findings. Evaluate the differences. The ability to repeat reported results is a critical part of the scientific process. Be able to defend or refute the results reported in the Chen et al . (1995) research article.


Study Guide

The two review articles by Weinberg and Bishop give a synthesis of the converging themes in understanding cancer. Read these articles first. What is the mechanism common to all forms of cancer? Next read the Chen et al. research article and two related follow up notes.

Be sure you understand the following terms and processes: Gene structure and function: transcription factor, exon, nuclear localization signal, intragenic mutation, zinc finger

Cell and tumor biology: steroid hormone receptor, neoplasm, tissue, immunocytochemistry, phosphorylation/dephosphorylation, metabolic labeling using radioisotopes, lymphocyte, epitope.


Definitions

Somatic and Germ Line Mutations - Read about the general properties of these mutations in the Principles of Genetics text.

Loss of Heterozygosity (LOH) - This term refers to the loss of the usual heterozygous combination of maternal and paternal alleles of genes at any given region of a chromosome. During tumor formation, one allele may be lost by a deletion or inactivated by a point mutation (perhaps induced by an environmental carcinogen). The second allele may be inactivated in a similar way or lost by mitotic recombination. When this happens, the repair systems in the cell try to repair the loss (often along with flanking regions of the chromosome) by a copying the corresponding region of the homologous chromosome (which may itself be defective).

Linkage analysis of kindreds - This type of analysis makes use of patterns of inheritance of markers throughout the genome. In linkage analysis, researchers determine whether any markers (already mapped genes, RFLPs, SSLPs) are consistently inherited along with the disease. If they do, they are said to cosegregate and the disease gene is assumed to lie somewhere in the area of the marker. (Note that this does not say the marker has anything to do with the disease.) It has been shown that BRCA1 is linked to more than half of all inherited breast cancers and 80% of families with both inherited breast and ovarian cancer.

Polyclonal antibodies are prepared by injecting an animal with an antigen (such as a protein) and isolating the antibody fraction from the animal's serum several weeks later. These consist of a heterogeneous population of antibodies, each directed against a different part of the antigen and produced by different cells in the animal. Monoclonal antibodies, on the other hand, are homogeneous; each is directed against just one specific part of the antigen and is the product of a single clone of cells. Monoclonal antibodies are made by fusing an antibody-producing lymphocyte from an immunized animal with a tumor cell and then growing a large population of these hybrid cells ("hybridomas") in culture. Remember from Unit I that every antibody molecule consists of two sets of domains which have specific functions (See The World of the Cell and Biology texts for more details). Be sure you know the molecular weights of the polypeptide chains that make up an antibody molecule.

Preimmune mouse serum is serum taken from a mouse prior to injecting it with antigen.

Site-specific refers to tissues and could be reworded as tissue-specific.


Introduction (Note that this report has a brief introductory paragraph as opposed to a full Introduction Section.)

    State in your own words why the authors conducted this study on BRCA1.

Results

Figure 1

  • Here is how the authors produced the antibodies they used: The GST expression vector contained the sequence for the C-terminus of the glutathione-S-transferase (GST) gene in addition to part of the BRCA1 gene and a bacterial promoter. It was put into the pGEX-2T prokaryotic expression system where it directed the synthesis of the recombinant (or "fusion") GST-BRCA1 protein. Next, the fusion protein was purified from the bacterial protein by running the preparation over a glutathione-sepharose column. The GST portion of the BRCA1-GST fusion protein binds to glutathione, the substrate for the GST enzyme. Pure BRCA1 protein was obtained by removing the GST portion of the BRCA1-GST fusion protein using a proteolytic enzyme that recognizes a site on the GST part. Thus, the authors could purify a large quantity of BRCA1 (or regions of BRCA1) in one step. They used this protein to generate antibodies.
  • HBL100 are human diploid breast epithelial cells which have been transformed with a virus and thus can grow in culture indefinitely.
  • The authors used radioactive 35S-methionine to trace proteins in cells. The cells were supplied with 35S-methionine, which was incorporated into newly synthesized proteins. This technique, called metabolic labeling, is very sensitive and can detect fewer than 1000 copies of a molecule in a sample.
  • Immunoprecipitation is one of the most useful immunochemical techniques. The immunoprecipitation procedure can be broken down into four steps:
    • labeling the antigen - in this report, BRCA1 is labeled with35S-methionine
    • lysis of the cells to release the antigen, BRCA1
    • formation of the antibody-antigen complexes
    • purification of the antibody-antigen complexes
  • 32P phosphoric acid was used as a source of radioactive phosphoryl group. The authors were trying to see if BRCA1 was post-translationally phosphorylated.
  • Breast cancer lines are breast epithelial cells grown in tissue culture. Usually, breast epithelial cell lines are derived directly from cancerous breast tissue. The tissue is treated with trypsin to release single cells, which are then put into tissue culture dishes containing growth medium. Cells adhere to the dish, begin to divide, and provide a source of material from one human. Often cancerous cells grown in culture are immortal, that is, they are able to grow forever in tissue culture. Immortality of cells grown in tissue culture is often, but not always, associated with the cells' ability to produce a tumor when injected into a mouse. The breast cancer cell lines analyzed in Figure 1B were isolated from different individuals. The same process was used to give the tumor cell lines derived from other tissues shown in Figure 1C.

    Why did the authors conduct a double immunoprecipitation with the BRCA1 polyclonal antibody?

    Two additional antibodies were used in the experiments found in Figure 1. In your opinion, was the use of these additional antibodies necessary?

Figure 2

  • Cell fractionation into nuclear, cytoplasmic, and membrane components is central to this study. Recall from doing this in Biocore 304 that the fractions are far from pure. What controls did the authors do to address the quality of their fractionation?
  • Indirect immunofluorescent staining was used extensively in Figure 2. Look up in Biology how researchers use immunofluorescence microscopy to determine the location of molecules in the cell.
  • The MDA-MB361 cell line was analyzed because it was derived from breast cancer cells that had metastasized to the brain. Cells that metastasize have undergone progression to invasive cancer as described Weinberg's article "How Cancer Arises."
  • A heterogeneous cell population is one that contains cells with different morphologies and/or different genotypes.
  • The pleura is a membrane enveloping the lung and lining the internal surface of the thoracic cavity. A malignant pleural effusion refers to fluid containing breast cancer cells. Normally, no more than 15 ml of clear fluid (devoid of cells) lubricates the pleural surface. Increased accumulation of pleural fluid can occur during breast cancer. This occurs because cells from advanced breast carcinoma frequently penetrate the thoracic wall and grow in the pleural fluid.
  • A common characteristic of some types of tumor cells is that they can divide without being attached to a surface (anchorage-independent growth). Such cells can grow in tissue culture medium without adhering to the tissue culture dish. This is termed grown in suspension.
  • A tumor that invades the underlying tissue and sheds cells into the blood or lymph is an advanced metastatic cancer.
  • A primary tumor is the initial tumor in the tissue of origin.
  • End-stage breast cancer refers to advanced metastatic breast cancer which ultimately is lethal because the metastatic tumors disrupt vital organs.
  • Stromal cells and lymphocytes from tumor refer to other cell types found in tissues. Breast tissue that contains a malignant tumor also has normal cells. Stroma is the supporting framework of an organ, such as the connective tissue, vessels, and nerves. It does not include the epithelial cells. Lymphocytes are white blood cells which mediate both humoral and cellular immune responses. For more information on lymphocytes, seeBiology.

    The authors used several controls in this experiment. List four controls and the experimental purpose of each.

    What data is conspicuously missing from Figure 2A?


Discussion (Note that this report has a brief discussion consisting of only two paragraphs)

    The authors conclude that BRCA 1 abnormalities are fundamental to both sporadic breast cancers and hereditary breast cancers. Do you agree? What is the evidence?

    Be able to explain parallels between BRCA1 and steroid hormone receptor molecules.


Technical Comment and Response Regarding the Research Paper

Scully, R., Ganesan, S., Brown, M., DeCaprio, J.A., Cannistra, S.A., Feunteun, J., Schnitt, S. Livingston, D.M. (1996) Technical Comment: Location of BRCA1 in Human Breast and Ovarian Cancer Cells. Science 272:123-125.

Chen, Y., Chen, P.-L., Riley, D.J., Lee, W.H., Allred, D.C., Osborne, C.K. (1996) Response: Location of BRCA1 in Human Breast and Ovarian Cancer Cells. Science 272:125-126.


Results

  • Fixation is required to prevent antigen leakage, permeabilize the cell to allow access of the antibody, keep the antigen in a form that can be recognized efficiently by the antibody, and maintain the cell structure. Fixation methods fall into two classes, organic solvents and cross-linking reagents. Organic solvents, such as alcohols and acetone, remove lipids and dehydrate the cells, precipitating the proteins on the cellular architecture. Cross-linking reagents form intermolecular bridges, normally through free amino groups, thus creating a network of linked antigens. Fixation in formaldehyde or glutaradehyde may mask or change some epitopes.

    What is the material at the bottom of the gel in Figure 1 of the Sculley et al., Technical Comment?

    Why aren't these same heavily stained bands seen in Figure 1 of the Chen et al., report? How do the techniques used in these two figures differ from each other?

    In their reply to the technical comments of Sculy and coworkers, how did Chen et al.., directly address the problems associated with cross-reactivity of the antibody reagents and fixation procedures?

    What additional information was learned about BRCA1 location/mislocation in breast cancer cells as a result of the two technical comment articles?


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