Molecular genetic analysis of the INK4a gene products and telomerase RNA component in vivo

Abstract

By dissociating complexes involving CDKs and preventing them from phosphorylating the Retinoblastoma protein, p16{dollar}\rm\sp{lcub}INK4a{rcub}{dollar} is believed to function as a growth suppressor regulating G1/S progression. In addition, since the p16{dollar}\rm\sp{lcub}INK4a{rcub}{dollar} gene is a common target for cytogenetic lesions in several human malignancies, it appears to be a candidate tumor suppressor. To confirm this hypothesis and study its in vivo functions, this locus has been disrupted in the mouse.;INK4a-deficient mice are viable and fertile, but exhibited spontaneous and carcinogen-induced tumors. Enhanced susceptibility to cancer is in accord with cell culture studies demonstrating a greater ease of immortalization and Ha-ras-induced transformation of primary INK4a-null fibroblasts. These findings fortify that the INK4a-locus suppresses neoplastic growth. INK4a-null mice also display a post-natal disorder characterized by progressive hepatosplenomegaly and extramedullary hematopoiesis in spleen, liver and lung. Most hematopoietic lineages appear to be affected with the most prominent abnormality in erythroid lineage. With advancing age, a subset of mice develop an acute expansion of B-lymphoid lineage cells. Thus INK4a proteins appear to regulate the proliferation and/or differentiation of hematopoietic lineages, and INK4a-null mice may serve as a model for age-related progressive leukemogenesis/lymphomagenesis.;Telomeres provide stability to chromosomes, and are synthesized by the RNP-DNA polymerase, telomerase. During development, telomere length shortens and telomerase is down-regulated and reactivated at late stages in tumor progression. Thus, telomerase is as a candidate target for cancer chemotherapy.;To study the effect of telomerase inhibition and in vivo function, we have generated mTR-null mice. mTR{dollar}\sp{lcub}-/-{rcub}{dollar} mice are viable, fertile and lack telomerase activity. Gross anatomy, histology, complete blood count profiles, peripheral smear, and Fluorescence-Activated-Cell-Sorting did not reveal abnormalities. Early generations of telomerase-deficient mice did not show a significant telomere-reduction. This lack of phenotype could be due to the unusually long telomeres in Mus musculus. Results of T-cell proliferation assay exhibited impaired responses to mitogens. Based upon these results, this will provide a model in which to assess key issues relating to cellular senescence, organ homeostasis and renewal, and cancer pathogenesis.