Use of phytohemagglutinin response to determine the extent that somatic cell mutation accounts for 6-thioguanine resistance of human blood mononuclear cells.

We investigated the likelihood that human peripheral blood mononuclear cells which incorporate tritiated thymidine in the presence of 6-thioguanine (6-TG) are mutants by comparing findings for cells from the same blood specimen cultured in the presence or absence of phytohemagglutinin (PHA). After 42 h in culture, autoradiography revealed that about 0.1% of cells from healthy control donors and 1% of cells from patients receiving chemotherapy for breast cancer labeled in the absence of PHA. About 5% of this number labeled when 6-TG was added to the medium. This high frequency of cells labeling in the presence of 6-TG suggested that many cells were resistant to 6-TG for reasons other than mutation at the HGPRT locus. Despite a labeling index of about 30% in the presence of PHA alone, the addition of PHA to cells cultured with 6-TG did not increase the labeling index over that observed with 6-TG alone. Also, in the presence of 6-TG there was a significant correlation between labeling indices for cells from the same blood specimen cultured with or without PHA. These findings suggest that the two conditions measured the same population of non-mutant cells. These studies reinforce observations made by the developers of this technique and other laboratories which indicate that refinements will be required before the assay can be used to study somatic cell mutation in human populations. Such refinements are underway in several laboratories; although not definitive, the approach and procedures used in this report which investigated the original assay procedure provide methods and criteria to judge whether modifications of assay techniques improve the quantification of in vivo human somatic cell mutation.