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Dissertation
Cloning, molecular characterization and altered expression of human twist during in vitro senescence of human diploid fibroblasts
Doctor of Philosophy (Ph.D.), Medical College of Pennsylvania and Hahnemann University
Oct 1995
DOI:
https://doi.org/10.17918/00007782
Abstract
Aging (senescence) is the occurrence of deleterious, cumulative, and apparently universal changes in living organisms that lead to an increased probability of morbidity and death. The limited proliferative potential of human diploid fibroblasts is considered as a model for this process, and this perception has provided the foundation for current approaches to the study of aging at the cellular level. The theories of cellular aging mechanism have been grouped into two distinctive categories: accumulation of random damages (extrinsic), and alteration of genetic control (intrinsic). Both may play a role both in in vivo aging and in the process of cellular aging. In our studies, we have emphasized on the possible role of altered genetic regulation during the cellular aging process. Several differentially expressed genes have been isolated from a subtracted library of young and senescent WI-38 human fetal lung-derived fibroblasts. I report here the cloning, sequencing and characterization of one of these differentially expressed genes, human twist. H-twist mRNA is overexpressed in young quiescent versus senescent human diploid fibroblasts. Twist genes have been characterized in different species including Drosophila, Xenopus, and mouse. They encode basic helix-loop DNA-binding transcription factors that play crucial roles in mesoderm development. The H-twist gene encodes a protein of 201 amino acids with 96% amino acid sequence identity to mouse twist, and 100% sequence conservation in the DNA-binding region among all species in which it has been characterized. The expression of human twistis primarily observed in mesoderm-derived cell lines and tissues. This expression patern is consistent with previous studies of the twist gene in Drosophila, Xenopus mouse, and suggests a fundamental role of the twist gene in mesoderm formation. I have also studied the pattern of H-twist expression as a function of in vitro lifespan in human diploid fibroblasts (HDF). H-twist is found to be preferentially expressed in young, Go cultures, and addition of fresh serum to these cultures results in a rapid repression of H-twist expression. This repression depends on the presence of growth factors, specifically EGF, and requires protein synthesis. Studies of mRNA stability, using actinomycin D or [alpha]-amanitin indicate a similar decay rate of H-twist mRNA in both young and senescent cells, suggesting that the differential expression of H-twist in young versus old is not primarily regulated at the level of possible role of mRNA turnover. Further exploration of H-twist on growth inhibition. This potential role of H-twist as a negative growth regulator suggests an important function in the control of fundamental processes such as cell proliferation, differentiation, senescence, and tumor formation.
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Details
- Title
- Cloning, molecular characterization and altered expression of human twist during in vitro senescence of human diploid fibroblasts
- Creators
- Sherry M. Wang
- Contributors
- Vincent J. Cristofalo (Advisor) - Drexel University, Medical College of Pennsylvania and Hahnemann University (1993-1996, 1998-2002)
- Awarding Institution
- Medical College of Pennsylvania and Hahnemann University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Medical College of Pennsylvania and Hahnemann University; Philadelphia, Pennsylvania
- Number of pages
- xii, 201 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Medical College of Pennsylvania and Hahnemann University (1993-1996, 1998-2002)
- Other Identifier
- 991021888736804721