Gene expression--Research--Methodology

Research into the chemical constituents of marine organisms is a relatively new
and growing field, given that much of the marine environment has only in the last half
century become accessible. Despite the strides that have been made in ocean exploration,
the marine biome has still been only barely explored. Much of the impetus for the
isolation and identification of chemical entities from marine organisms has been related
to their potential as medicines. For a number of reasons, including synthetic efficiency,
this has meant that many marine compounds isolated and studied have been small
molecules. In recent years, large strides have been made in techniques for the isolation
and identification of biological macromolecules, especially proteins. The understanding
of these molecules, and their relationship to the biochemical processes of the marine
organisms from which they have been isolated, is important not only because of the
potential for this information to help in the synthesis of medicines, but because it may help in the recognition of processes that affect the very viability of marine organisms
increasingly exposed to anthropogenic threats to their environment. This work consists of
four related studies involving the development of methods for the separation and
identification of proteins from a number or gorgon ian species. Chromatographic and gel
based methods were used to isolate an elisabethatriene synthase (ELS) which shows
promise as a biotechnological tool for the production or pseudopterosins. A number of
gorgonians were screened for the presence of proteins that might have antimicrobial
properties, and several organisms were identified that might be of interest in this context.
Two-dimensional gel electrophoresis methods were then developed to allow the
construction of gel maps for the azooxanthellate gorgonian Leptogorgia minimata and the
xoozanthellae gorgonian Pseudopterogorgia e/isahethae, the separated proteins were
digested and analyzed by LC/MSMS. and the information generated was used to examine
the proteome of the organism for functional and phylogenetic relationships.
Finally, the gorgonian Euniceafi<sca was exposed to several environmental stressors in
the laboratory. and the two-dimensional proteomic methods developed were used to
examine the effect of the stressors on the organism .

A Down's Syndrome related Single Minded 2 gene (SIM2), previously known to be
associated with Trisomy 21 was predicted by bioinformatics to be colon cancer specific.
In previous work from the laboratory using a patient tissue repository, an isoform of this
gene, short form (SIM2-s) was shown to be colon cancer specific. Inhibition of SIM2-s
expression by antisense technology resulted in cancer-cell specific apoptosis within 24
hours. Microarray-based gene expression profiling of the antisense-treated colon cancer
cells provided a fingerprint of genes involving key cell cycle, apoptosis, DNA damage
and differentiation genes. Taking hints from the microarray database, experiments were
initiated to decipher the molecular mechanism underlying the cancer specific function of
the SIM2-s gene. Using an isogenic cell system, apoptosis was found to be dependent
on DNA damage and repair gene, GADD45-a. Further, key pathways including p38 MAP
kinase (MAPK) and specific caspases were essential for apoptosis. Programmed cell
death was not dependant on cell cycle and was preceded by the induction of terminal
differentiation. To clarify whether SIM2-s function is a critical determinant of differentiation, stable transfectants of SIM2-s were established in a murine adipocytic
cell line (3T3-L 1 ). SIM2-s overexpression caused a pronounced block of differentiation
of the pre-adipocytes into mature adipocytes. A study of the differentiation pathway in
3T3-L 1 cells suggested that this block occurs early on in the cascade. These results
supported the starting premise that SIM2-s is a critical mediator of cell differentiation. To
clarify whether the SIM2-s gene has transforming potential, the SIM2-s gene was
overexpressed in the NIH3T3 murine fibroblast cell line. The cells expressing the human
SIM2-s gene exhibited shorter doubling time, abrogation of growth serum requirement,
greater cell number at saturation density and focus formation. In vivo tumorigenicity
assays showed tumor formation with long latency. These results provide strong evidence
for the role of SIM2-s gene in tumor cell growth and differentiation, and validate drug
therapy use for the gene.

Harnessing the human genome using bioinformatics lead to the discovery of a highly cancer-selective gene, Single Minded 2 gene (SIM2). An isoform of the SIM2 gene, the short-form (SIM2-s), was shown to be specific to colon, pancreas, and prostate tumors. Antisense inhibition of SIM2-s in a colon carcinoma derived cell line (RKO) caused inhibition of gene expression, growth inhibition and apoptosis in vitro and in nude mice tumorigenicity models. To understand the mechanism of Sim2-s antisense, the antisense treated RKO colon cancer cells were monitored for genome wide expression using Affymetrix GeneChipRTM technology. A list of apoptosis related genes was generated using GeneSpringRTM software. Select GeneChip RTM output was validated by Quantitative RT-PCR. Relevance of a key gene, Growth arrest and DNA damage inducible (GADD45a), in the SIM2-s pathway was established. These results will provide a basis for the future experiments to understand the mechanism underlying Sim2-s activation in specific tumors.