Analysis of molecular changes upon immortalization of SV40-transformed human fibroblasts.
Item
-
Title
-
Analysis of molecular changes upon immortalization of SV40-transformed human fibroblasts.
-
Identifier
-
AAI9510700
-
identifier
-
9510700
-
Creator
-
Pardinas, Jose Ramon.
-
Contributor
-
Adviser: Harvey Leon Ozer
-
Date
-
1994
-
Language
-
English
-
Publisher
-
City University of New York.
-
Subject
-
Chemistry, Biochemistry | Biology, Molecular | Biology, Genetics
-
Abstract
-
Normal human diploid fibroblasts (HDF) undergo a pattern of proliferative decline in tissue culture which, by analogy to aging in the organism, has been termed cellular senescence. HDF transformed by the DNA virus SV40 have an extended lifespan, but inevitably succumb to a pattern of growth cessation designated "crisis". As a rare event, SV40-transformed fibroblast (SV/HF) populations may give rise to cell variants which are immortal. I have examined the organization and expression of viral and cellular sequences in preimmortal and immortal SV/HF in an effort to understand the molecular basis for immortalization. While rearrangements were not detected in the SV40 integrant, the immortal cells show a consistent 2-3 fold higher level of hybridization to the SV40 probe than the preimmortal progenitor. Examination of cellular DNA organization revealed several interesting changes. First, nonrandom alterations in the long arm of chromosome 6 (6q) were noted in association with the emergence of immortal cells. I have demonstrated a 2-fold reduction in the copy number of DNA sequences distal to 6q21 in the immortalized transformants consistent with the presence at this locus of a growth suppressor whose complete loss (or dosage reduction) is critical for immortalization. Secondly, since loss of telomeric DNA has been implicated in the proliferative decline of cellular senescence, telomere lengths were determined at various times during the lifespan of normal HDF, as well as of multiple matched sets of preimmortal and immortal SV/HF. In our cell lines telomeres also display progressive shortening with cumulative population doublings of normal HDF and, furthermore, are found to continue to decrease during the extended lifespan of preimmortal SV/HF. However, telomere lengths increase and stabilize as SV/HF become immortal. These results argue against a causal role for telomere shortening in cellular senescence, and imply that immortalization induces or is partly dependent on activation of telomere maintenance mechanisms.;In order to identify mRNA species that are differentially expressed in the preimmortal and immortal SV/HF, I prepared a preimmortal cDNA library in {dollar}\lambda{dollar} gt10 and analyzed it by subtractive hybridization. A number of cDNAs have been isolated representing mRNAs which are overexpressed in either the preimmortal or the immortal cells. Partial sequencing indicates that most of these (11 out of 14) correspond to novel genes.
-
Type
-
dissertation
-
Source
-
PQT Legacy CUNY.xlsx
-
degree
-
Ph.D.
