Evidence of a functional receptor in Prostate cancer cells (LnCaP)

By: Saphir NiakadieMentor: Dr. Anthony DePass

Long Island University, Brooklyn Campus

Introduction

• Apoptosis is a naturally occurring event that can be triggered by a number of stimuli such as toxins, oxidative stress, calcium influxes and the activation of death receptors.

Introduction con’t

• It has been concluded that apoptosis is orchestrated by calcium levels (Mattson, 2003), and we therefore have hypothesized that during different stages of apoptosis, calcium levels within LnCaP cells should be depleted and external levels should increase

Introduction con’t• During the course of the summer, I will be working along

side Dr. Anthony DePass and Carleta Williams investigating the role of ryanodine receptor in prostate cancer (LnCaP) cells that has been infected with Adenovirus MDA-7 to induce apoptosis.

Background Information

• Ryanodine receptors are a family of intracellular calcium channels that aid in the regulation of calcium levels inside cells.

• There are three known isoforms of the Ryanodine receptor:

1.RyR1 found in Skeletal muscle2.RyR2 found in cardiac muscle3.RyR3 found in brain tissue

Background Information• Melanoma differentiated associated gene-7 (MDA-7) is a

tumor supressor gene. Studies have shown that transfer using an adenovirus exhibits minimal damage to normal cells.

Methods and Procedure• In order to establish evidence that the

ryanodine receptor is present in LnCaP cells after treatment with Ad. MDA-7, RT-PCR and Western Blot analysis of samples extracted at various time points will be assessed.

• Calcium measurements will also be taken from cells infected with the virus.

Methods and ProceduresAdenovirus infection and RNA extraction

• LnCaP cells were cultured in RPMI medium and supplemented with 5% CO2.

• Cells were plated in 100mm petri dishes and infected with Ad. MDA-7.

• RNA was were extracted from cells, 24, 48 and 72 hours after initial infection

Methods and ProceduresRT-PCR

• Reverse Transcriptase – Polymerase Chain Reactions (RT-PCR) will be done to show evidence of a functional ryanodine receptor from mRNA extracts.

• RT takes the RNA and transcribes it into DNA, then PCR uses the very small segment of DNA and make millions of copies.

Methods and ProceduresProtein assay and Western Blot analysis

• Protein was also extracted from LnCaP cells after 24, 48 and 72 hours from initial infection.

• A polyacrylamide gel will be used in the western blotting procedure, then transferred to a nitrocellulose membrane for easier handling.

Discussion• A previous study has shown that when using RT-PCR two isoforms

RyR1 and RyR2 of the ryanodine receptor were detected in LnCaP cells (Mariot et al, 2000).

Expected results• For the RT-PCR procedure we expect an amplified product

of about 554 base pairs (bp) and 476 bp indicating the ryanodine receptor is fully functioning.

Expected results• For the Western Blot procedure, we expect to find bands around the 565

kDa marker. This will prove that ryanodine express proteins in LnCaP cells which then shows evidence that the receptor is existent.

References• Mark P Mattson and Sic L .Chan 2003. Calcium orchestrates apoptosis. Nature

cell biology volume 5 number 12

• Pascal Mariot and Natalia Prevarskya et al 2000. Evidence of functional Ryanodine Receptor Involved in Apoptosis of Prostate cells. The Prostate 43:205-214

• Zhaozhong Su and Luni Emdad et al 2005. Unique Aspect of Mda7/IL-24 antitumor bystander activity : establishing a role for secretion of MDA-7/IL24protein by normal cells.

• http://medix.marshall.edu/~sergeev/images/apoptosis.jpg

• http://bricker.tcnj.edu/tech/ltle1/plus.jpg

• http://www.scbt.com/

Acknowlegdments

• Dr. Anthony DePass

• Carleta Williams

• Dr. Sat

• Harlem Children Society

• MSKCC

THANK YOU!!!