Cloning of Senescence Genes in Mammary Epithelial Cells

Institution: Stanford University
Investigator(s): Hong Zhang, Ph.D. -
Award Cycle: 1999 (Cycle V) Grant #: 5FB-0067 Award: $75,034
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point

Initial Award Abstract (1999)
Cancer is a group of diseases resulting from changes in genes that control cell growth and behavior. To become malignantly transformed, a normal human cell needs to acquire genetic changes in multiple critical genes. Alterations in many genes that control cell growth and death can contribute to neoplastic transformation. Normal primary human cells have limited life spans, and they can only proliferate for a limited number of time before entering a stage called cellular senescence. One of the most common changes in cancer cells is that they have lost the ability to undergo senescence and can divide indefinitely becoming "immortal". Understanding of the genetic basis for senescence in cells of the human breast will enable the development of new therapeutic approaches and new management measures for cancers. However, the genetic basis for cellular senescence is not well understood.

The overall goal of the proposed study is to identify the genes that control cellular senescence in human mammary epithelial cells, and study the functions of isolated senescence genes. The proposed study will employ a new genetic procedure that was recently developed in my mentorís laboratory. This procedure allows the identification of previously unknown genes that play a crucial role in the control of cellular senescence. Identification of genes and genetic pathways of cellular senescence will provide therapeutic targets for restoration of cellular senescence in breast cancer cells. Other translational potential of the proposed work includes the design of epidemiological approaches for accessing the genetic susceptibility of individuals to develop breast cancer and the creation of prognostic tests for managing breast cancer.

Final Report (2001)
Normal body cells have only a limited capacity for growth in culture and soon stop growing, entering a state called replicative senescence. The inability of such senescent cells to continue to divide has led people to suggest that replicative senescence may be a tumor suppression mechanism that prevents changes in normal cells that lead to cancer. It also has been suggested that the inability of cells to divide that occurs during replicative senescence represents aging at the cellular level. There is evidence that senescence is genetically determined and the signal feature of replicative senescence is telomere shortening. The overall objective of this grant is to study the genetic basis of replicative senescence. The approaches that I have taken include both Genetics and reverse Genetics.

Reverse Genetics approach: I used high-density cDNA microarrays to investigate the nature of alterations in gene expression during telomere-dependent replicative senescence in fibroblasts and HMECs, and to compare gene expression during senescence with expression during physiologically-induced growth arrest (i.e. quiescence). I identified genes whose altered expression was unique to, or common to, senescence or quiescence in fibroblasts and HMECs, and defined gene expression fingerprints characteristic of these growth arrest states. Multiple alterations believed to reflect senescence were found instead to be non-specific concomitants of growth arrest. Remarkably, there was no similarity at all between telomere-dependent senescence-specific gene regulation in fibroblasts and HMECs, indicating that the replicative senescence phenotype is extensively polymorphic at the level of gene expression.

Genetics approach: I used a new generation of random homozygous knockout (RHKO) vectors to identify genes involved in senescence in human mammary epithelial cells. The initial phenotype selection of ~25,000 independent RHKO clones yielded one clone that has extended life span. Additional RHKO libraries are being generated and the phenotype selection will be carried out. Two genes whose expression is specifically altered during senescence are currently under investigation for their role in replicative senescence. Preliminary study indicates that overexpression of one gene inhibits cell growth and induces a pattern of gene expression characteristic of senescence.

The senescence-specific alterations in gene expression I identified in human fibroblasts provide a fingerprint to distinguish senescence from other growth arrest phenotypes. This senescence fingerprint potentially provides a tool for analyzing the causative role of specific genes, and also of non-genetic factors, in senescence. My finding that the gene expression profile of senescence in human mammary epithelial cells is profoundly different from that of human fibroblasts indicates that senescence is a polymorphic event rather than being commonly viewed as a singular biological phenomenon.