Quantitation of Go and G4 Phase Cells in Primary Carcinomas Antibody to M1 Subunit of Ribonucleotide Reductase Shows G. Phase Restriction Point Block

Human cancers have an apparent low growth fraction, the bulk of cells presumed to being out of cycle in a Go quiescent state due to the inability in the past to distinguish Go from G1 cells. The allosteric Ml subunit of ribonucleotide reductase (Ml-RR) is constitutively expressed by cycling cells (i.e., G1, S, G2-M). It is acquired during transition from Go to GI, lost during exit to Go and thus distinguishes Go from G1 cells. To estimate the proportion of Go and G1 cells in primary human breast (n = 5) and colorectal (n = 12) adenocarcinomas, we used both analytical DNA flow cytometry (ADFC) and im-munoperoxidase staining of sections with the monoclonal anti-body to MI-RR (MAb MI-RR). ADFC of fresh tumors re-vealed a low percentage of cells in the S phase (4.0±3.4%) but immunoperoxidase staining for Ml-RR revealed an unexpect-edly high proportion of positive cells (52.4±12.7%) in the GI, S, G2-M phases indicating a high G1 content of primary human tumors. Thus, human cancers are blocked in transition in G1 and are not predominantly in a Go or quiescent differentiated state. This block was interpreted to mean that human cancers are responding to putative regulatory events at a restriction point in the GI phase, such as relative growth factor deficiency, density inhibition, antiproliferative cytokines, or gene prod-ucts. Using flow cytometry for both DNA and MI-RR content we found that human colon cancer cell lines arrest in the G1 but not Go phase upon serum deprivation or density inhibition. Simi-larly, human breast cancer cell lines are arrested in G1 but not Go phase by medroxyprogesterone acetate (MPA) or tamoxifen exposure. These findings match our in situ observations, and support the concept of a restriction point block in primary hu