Dr. Richard J. Albertini
Genetics Laboratory
32 N. Prospect St.
Burlington, VT 05401-0508
Phone: 802-656-8866
Fax: 802-656-8333
Email: ralberti@moose.uvm.edu
Projects are approved by an IRB located at: University of Vermont.
The approving IRB operates under a Multiple Project Assurance (MPA) recognized by DOE or by the Department of Health and Human Services (HHS).
MPA number of the IRB: M-1375
Number of Human Subjects Projects reported: 1
Project Identifier: UVT-83-DE-FG02-87ER60502
Project Title:
The Development of In Vitro Mutagenicity Testing Systems Using T-Lymphocytes
Principle Investigator:
Dr. Richard J. Albertini
Principle Investigator's Institution: University of Vermont
Project started in: 1983
Project Funding Information:
Project received funding in Fiscal Year 1995.
Project used human subjects in Fiscal Year 1995.
Funding Sources:
Project does not involve use of multiple protocols/subprojects.
IRB Review:
Type of Review: Full Board
Most Recent Approval: July 07, 1995
IRB Approval Number: CHRMS 95-237
Number of Human Subjects in the Last Reporting Period for this Project: 140
(Reporting periods vary.)
Type of Human Subjects Involvement:
a. The overall goals of the research program of the Vermont Cancer Center Genetics Laboratory are to understand the causes, mechanisms, and consequences of somatic mutations in humans and to use this understanding to identify individuals at risk for deleterious health effects resulting from environmental mutagens/carcinogens. In this context, the theme of this research program has been mutation induction in human T-lymphocytes. The emphasis has been on using studies of in vitro mutations in T-lymphocytes as tools for the experimental analyses of in vivo observations. Our goal in this proposal is to define the genetic bases of mutator phenotypes arising in vivo in non-malignant cells. In particular, the sub-projects are to discover and characterize spontaneous/induced molecular mutational spectra in the hypoxanthine phosphoribosyltransferase (hprt) gene in human colon cancer cells; to use hprt mutation in T-cells to identify hypermutable clones among the hprt mutants arising in vivo in individuals constitutionally heterozygous for functionally relevant mutation in a mismatch repair gene; to use hprt mutation in T-cells to identify hypermutable clones among the hprt mutants arising in vitro following exposures of lymphocytes from mismatch repair constitutional heterozygotes to model mutagens; to characterize the hypermutable T-cell clones so obtained as to molecular spectra, in vitro hypersensitivity to killing, in vitro mutability, repair capacity, and the specific mutation in the inherited allele; to develop a system for direct selection of mismatch repair deficient T-cells arising in vivo in humans; and to identify nutrients or other agents that reduce the mutability of hypermutable cells.
b. The method used to study individuals and cells is the hprt clonal assay. This requires a blood sample. T-lymphocytes are isolated from the blood sample and the number of mutant lymphocytes determined by limiting dilution cloning. Molecular analysis of the mutations in the T-lymphocytes is then performed on the mutant lymphocytes. For some experiments, isolated lymphocytes are treated with radiation or chemicals in vitro. For some individuals, some data on age, smoking, and health history are collected.
c. Some individuals are cancer patients and have had chemotherapy or radiotherapy; however, this was not administered under this proposal. These patients were only studied because they had already received therapy. Some individuals have been studied because of radon exposure in their homes.
d.
1. In vivo hprt T-lymphocyte clonal assay informed consent is obtained and then blood samples collected in heparin tubes. The mononuclear cell layer is isolated and washed twice with saline. Cells are plated at limiting dilution in 96-well (round bottom) microtiter dishes at 1, 2, 5, and 10 cells/well in the absence of TG and at 1-3 x 10e4 cells/well in the presence of 10 micromolar (uM), thioguanine (TG), T-cell growth factor (TCGF), phytohemagglutinin (PHA), and accessory cells. The microtiter dishes are incubated for 10-16 days and growing colonies determined by use of an inverted phase contrast microscope. Wells are scored for colony growth on days 10-14. Mutant and wildtype colonies in positive wells are isolated, propagated and stored for molecular analysis. Mutant frequencies are calculated based on Poisson statistics as the ratio of the cloning efficiency (CE) in the selective 6-thioguanine medium divided by the CE in non-selection medium. CE are based on the fraction of wells with positive cell growth (CE = -ln Po/N, where Po is the fraction of wells without cell growth and N is the number of cells seeded into wells). The ratio of CE in the presence or absence of 6-thioguanine defines the mutant frequency.
In vitro assays for induced hprt mutations exposed human T-lymphocytes The in vitro assays employ either fresh or cryopreserved mononuclear (MNC) and are cultured in the same medium as described for the in vivo assay. The cells are exposed before mitogen stimulation, i.e., while in Go phase, in order to more closely approximate the in vivo situation. After exposure, cells are incubated with 1 u/ml PHA for 36-40 hours to achieve mitogen stimulation. The stimulated cells are plated as for the in vivo assay to determine the relative survival and the pre-existing mutant frequency. Phenotypic expression is accomplished by two methods. The first is through subculture for expression and the second is through in situ expression. The latter method is primarily used to isolate mutant clones with independent mutations for molecular analysis.
2. There are no risks except bruising and a very minor risk of infection at the site of blood draw.