| Abstract |
 Studies in the laboratory of Dorothy Teegarden focus on Vitamin D, which is associated with a reduced incidence of several cancers, including prostate, colon and breast cancer. Dr. Teegarden is investigating the role and the mechanism of Vitamin D metabolites in the growth of cells and programmed cell death (PCD or apoptosis). Cell models for multistage carcinogenesis are used to investigate these mediators. Studies in her laboratory have shown that 1,25 dihydroxyvitamin D inhibits apoptosis in "normal", untransformed cells. On the other hand, 1,25 dihydroxyvitamin D does not protect C3H10T1/2 cells transfected with the ras oncogene, a mutation found in approximately 30% of tumors, from apoptosis. Currently, the mechanism by which 1,25 dihydroxyvitamin D regulates hypoxia inducible factor is being investigated in breast cell model systems. In addition, the signaling pathways and the role of the nuclear vitamin D receptor mediating its effects are being explored.
Pharmacogenomic analysis is under the control of Dr. James Klaunig at Indiana University, Bloomington. The Vitamin D SNPs are included on a single chip with the oxidative stress and DNA repair enzymes.
With respect to colon cancer, Vitamin D status has been shown to exhibit preventive activity against colon cancer in cell, animal and human studies. Vitamin D, presumably via Vitamin D receptor-mediated effects on gene expression, results in antiproliferative effects in colon cancer cell lines and primary cultures from human colon tissue. Several lines of evidence also support a role for oxidative stress in disease progression. A lower risk of colorectal cancer has been noted following high antioxidant intake. In addition, antioxidant enzyme activities were decreased, and the extent of oxidative damage was increased in colon cancer tissue compared with normal tissue. Further, reactive oxygen species were shown to cause DNA damage in genes that are commonly mutated in colorectal tumors. Interestingly, oxidative stress induction and vitamin D status appear to be associated, as it was shown that oxidative DNA damage levels and cell proliferation were increased in colon tissue from mice lacking the vitamin D receptor. These results suggest that chemoprevention strategies involving both antioxidant and vitamin D supplementation may be useful for preventing colon carcinogenesis.
Pharmacogenomic Analysis
The pharmacogenomic analysis encompasses a comprehensive approach to understanding the relationship between known polymorphisms in critical genes associated with regulation of oxidative stress and damage, as well as the potential interaction of vitamin D status (including polymorphisms in vitamin D related genes), or other factors that may influence the development of colon carcinogenesis (intervention dietary modifications, etc). This analysis will contribute information for the statistical modeling that will identify molecular signatures that define potential susceptibility factors for colorectal development such that individuals at increased risk can be identified. This information may ultimately result in reduced risk for colon cancer development through the establishment of targeted intervention strategies aimed at preventing or delaying disease onset.
The following analyses will be performed on DNA extracted from white blood cells isolated from whole blood collected from colorectal cancer patients. SNP genotyping for genes involved in oxidative stress (enzymatic production and removal), vitamin D status, and epigenetic methylation; 2) measurement of oxidative stress parameters (antioxidant capacity, oxidative DNA damage and oxidative repair enzymes); and 3) quantification of vitamin D status. In addition, dietary records of colorectal cancer patients will be collected by the IUSCC clinical team, analyzed and correlated with the SNP and biological analyses.
Vitamin D Status Analysis
Vitamin D sample preparation, instrument analysis, and data post-processing are carried out at Intelimmune, L.L.C. (info@intelimmune.com) in the laboratory of Bo Xie, Ph.D, Director of Analytical Services.
3000 Kent Avenue,
Room B2-900,
Research Park
West Lafayette, IN 47906.
phone: 765-412-5850
 Instrumentation at Intelimmune includes the Shimadzu UPLC system and the Applied Biosystems 4000 QTRAP. The AB QTRAP, a Triple Quadrupole LC/MS/MS System, is a robust, highly sensitive instrument that provides advanced structure identification to accurately detect the masses of both molecular and product ions. Sample preparation and instrument setup protocols will be made available here in the near future. The AB 4000 QTRAP generates WIFF files during sample analysis (about 30 samples at a time), which are converted converted to Excel spreadsheets and then to CSV files. The files contain vitamin D metabolite data used as input for analysis in the laboratory of Dr. Teegarden. The dataset workflow is shown here.
Intelimmune proprietary assay protocol for the vitamin D metabolites 25 hydroxy-vitamin D2 and D3 is fully validated by comparison with reference values supplied by the National Institute of Standards (NIST) for SRM 972. See their standards reference material Standard Reference Material (SRM) 972 Vitamin D in Human Serum in powerpoint format.
The metabolites for Vitamin D analysis include:
- 24,25(OH)2VD3 and 24,25(OH)2VD2
- 1,25(OH)2VD3 and 1,25(OH)2VD2
- 25OHVD3 and 25OHVD2
- VD3 and VD2
Quantitative capabilities (serum/plasma matrix):
- Lower limit of quantification: 10 pg/mL
- Range: 10 pg/mL to 200 ng/mL
- Alternative matrices possible
An example matrix identifying accuracy and precision is shown here.
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