Related Experiment Videos
Detection of PDGF-2 homodimers in human tumor cells
H Igarashi1, C D Rao, M Siroff
1Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
Abstract:
The v-sis oncogene encodes a protein structurally and functionally related to human platelet-derived growth factor (PDGF). In the present studies, we show that the primary translational product of the human sis proto-oncogene is a 26-kd protein, p26c-sis. This product is processed to yield a disulfide-linked homodimer, p56c-sis, which is further processed to a 35,000-dalton dimer, p35c-sis. Like the v-sis gene product, the PDGF-2 precursor undergoes N-linked glycosylation, implying its processing through the endoplasmic reticulum. The PDGF-2 product was shown to possess functional properties of PDGF. Whereas lysates of control COS-1 cells lacked mitogenic activity, lysates of COS-1 cells transfected with a c-sis/PDGF-2 expression vector specifically stimulated DNA synthesis of quiescent fibroblasts. Moreover, this activity was completely inhibitable by PDGF antibody. Identical forms of the sis/PDGF-2 product were identified in human tumor cells that expressed c-sis/PDGF-2 transcripts. These proteins were shown to be specifically associated with the membrane component of the tumor cells and were not detectably secreted into the culture medium. These findings support the concept that expression of the sis/PDGF-2 product in human cells responsive to its proliferative actions can be an important step in the processes leading to malignancy.
Insights
The human sis proto-oncogene produces a protein similar to platelet-derived growth factor (PDGF). This protein stimulates cell growth and is found in tumor cells, suggesting a role in malignancy.
Area of Science:
- Oncogene research
- Molecular biology
- Cellular signaling
Background:
- The v-sis oncogene product is related to human platelet-derived growth factor (PDGF).
- Understanding the human sis proto-oncogene's protein products and their function is crucial for cancer research.
Purpose of the Study:
- To characterize the translational products of the human sis proto-oncogene.
- To investigate the functional properties and cellular localization of the sis/PDGF-2 product.
- To determine the role of the sis/PDGF-2 product in human malignancy.
Main Methods:
- Transfection of COS-1 cells with a c-sis/PDGF-2 expression vector.
- Analysis of protein products, including molecular weight and dimerization.
- Assay of mitogenic activity using quiescent fibroblasts.
- Immunoprecipitation and Western blotting using PDGF antibody.
- Identification of sis/PDGF-2 products in human tumor cells.
Main Results:
- The primary product of the human sis proto-oncogene is p26c-sis, processed into dimers p56c-sis and p35c-sis.
- The PDGF-2 precursor undergoes N-linked glycosylation, indicating endoplasmic reticulum processing.
- Transfected COS-1 cells expressing c-sis/PDGF-2 showed PDGF-like mitogenic activity, inhibitable by PDGF antibody.
- sis/PDGF-2 products were found associated with the membrane of human tumor cells and not secreted.
Conclusions:
- The sis/PDGF-2 product exhibits functional PDGF-like mitogenic activity.
- Expression of the sis/PDGF-2 product in responsive human cells may contribute to the development of malignancy.
- The membrane-associated localization in tumor cells suggests autocrine or paracrine signaling in cancer progression.