• Overview
• Degrees & Areas of Study
  • Core Program
  • Majors
    • Biology Major
    • Education Major
    • English Language and Literature Major
    • History Major
    • Interdisciplinary Major
    • Religion Major
  • Certification in Secondary Education
  • Associate in Arts Degree
• Experiential Learning
• Faculty
• Catalog and Course Information
• Support
• Academic Forms & Downloads
• Faculty Resources

<<Back

Summer Internship at Fox Chase Cancer Center 2002

By Johanna Smith

The lifecycle of the retrovirus HIV is well understood. The virus enters the cell by fusing with certain receptors present on the target cell membrane, the viral RNA reversely transcribes to DNA via the viral protein, reverse transcriptase, and then this viral DNA can be integrated into the host cell’s nuclear DNA. However, there are a lot of unanswered questions about the process of integration the retroviral lifecycle. We know that host cellular repair mechanisms are essential for this involved step but exactly what and how this happens are still in uncertain. After this involved step, the provirus, or integrated virus, can produce new virions by taking advantage of host cell mechanisms to transcribe and translate the necessary proteins and RNA molecules to make a new virus. The necessary components of a complete viral particle include two RNA molecules, reverse transcriptase, integrase, and several other virus proteins. These are then packaged in the cytoplasm and released from the host cell by budding off of the host cell membrane. The newly formed particles can move on to new victims, or host cells.

DNA damage is constantly occurring in our cells. We have evolved complex mechanisms in order to repair our genetic material when damage takes place. When this damage arises in a healthy cell, cell cycle checkpoints are activated which stall the process of cell division until the DNA can be repaired. If the damage is too great, the cell may kill itself by a programmed cellular suicide called apoptosis. When DNA damage cannot be repaired properly and the cell cycle continues, this is when uncontrollable cell propagation is cancerous. Up until recently, in order for cellular repair and checkpoint mechanisms to be studied, ionizing radiation was the only method used for inducing DNA damage. However, with our more advanced understanding of retroviral integration, we can now study this process and learn not only about retroviruses, but how DNA is repaired in all cells when damage takes place. Integration provides a more “gentle” approach to inducing DNA breaks.

BRCA1 is a protein that is not fully understood. However, mutations in this protein have been accountable for about 85% of inheritable breast cancer. Evidence is also showing relevance of BRCA1 in DNA repair. Currently I am using cells that are deficient in this protein (BRCA1-/-) to see whether or not it is important in retroviral integration.

I have learned to make a mutated form of HIV by transfecting producer cells with three plasmids which code for the “backbone” of the HIV virus, a lac Z reporter gene, and a virus envelope protein that is compatible to the receptors on the host cells which will be infected with the virus. This form of the HIV virus can infect cells, but once it infects and integrates, the process cannot continue. This is because it does not contain a packaging sequence, which is required to round up all of the necessary components in the cytoplasm in order for new viruses to be made and released from the cell. So this is a safe form of HIV that I can work with.

Once I collect the virus from these producer cells, I can infect the host cells that I am using to carry out the experiment. The experimental host cells that are of interest are from a cell line which is deficient in BRCA1. In order to determine if retroviral integration has occurred, I do a beta-galactosidase assay which utilizes the lac Z reporter gene. The lac Z gene codes an enzyme (beta-galactosidase) which is implicated in the breakdown of lactose to the form of gluecose and galactose. By applying a staining solution that contains X-gal, I can determine if integration has successfully occurred. This is because X-gal is broken down by Beta-galactosidase to a product that is deep blue. So the cells that turn blue contain the HIV genetic material which has been integrated into their DNA. Cells that have not been infected with the virus remain white. The difference in color can be easily determined under a microscope, and I count the number of blue cells. By doing so I can compare the number of infected cells which are deficient in BRCA1 to another cell line which contains the wild type form of BRCA1.

Johanna A. Smith graduated with a B.S. from the Bryn Athyn College Biology Major in 2004. The summer of 2002 she did an internship with Dr. Rene Daniel and Anna Marie Skalka at the Fox Chase Cancer Center Institute for Cancer Research.