Assessment of cell proliferation by AgNOR scores and Ki-67 labeling indices and a comparison with potential doubling times

Abstract

The potential doubling time (T[pot]) has been proposed as a pretreatment estimator of intratreatment tumor proliferative capacity. The assay has several limitations, however, including the inability to discriminate between host and tumor cells in diploid tumors and the need for prospective labeling of patients with a halogenated pyrimidine. Often a separate biopsy is needed as well. In this study, Ki-67 immunostaining and AgNOR silver staining were examined as possible alternative methods by comparing them with the T[pot] in an in vitro system using two different cell lines under varying growth conditions. Using CaSki cells, nutritional status was varied to alter proliferation rates, and a strong correlation was found between Ki-67 labeling index and AgNOR scores (r = 0.927, P = 0.0003). Both Ki-67 labeling index and AgNOR scores had statistically significant inverse correlations with cell doubling time (T[d]), length of S phase (T[s]), and T(pot), as well as a positive correlation with iododeoxyuridine (IdUrd) labeling index. As an alternative method of inhibiting proliferation, 4-hydroxytamoxifen (4OH-TAM), the potent anti-estrogenic metabolite of tamoxifen, was used to inhibit the proliferation of estrogen-dependent MCF-7 cells. Treatment with the anti-estrogen resulted in decreased Ki-67 labeling index and AgNOR scores of MCF-7 cells, correlating with a decrease in IdUrd labeling index and inversely correlating with T(pot). The significant correlations among Ki-67 labeling index, AgNOR scores, and these other measures of proliferation suggest their potential value as indicators of proliferative activity. Retrospective markers, such as Ki-67 labeling index or AgNOR score, may be particularly attractive clinically in that prospective labeling of patients with halogenated pyrimidines could be avoided; however, extensive clinical testing will be required before the clinical usefulness of these markers can be established.