Discrimination of human leukemia subtypes by flow cytometric analysis of cellular DNA and RNA

Abstract A newly developed flow cytometry technique for simultaneous measurements of three features of individual cells--DNA, RNA, and nuclear diameter--using acridine orange as a fluorescent metachromatic dye, has been applied to cell-cycle analysis. DNA stemline determination, and to classification of 102 cases of human leukemias in adults. Acute lymphoblastic leukemia (L1–2) was characterized by moderately increased RNA of G0/1 cells as compared to normal lymphocytes; acute nonlymphoblastic leukemia (M 1–5) by very high RNA of G0/G1 cells. Both had either diploid or aneuploid DNA stemlines. Chronic lymphocytic leukemia showed diploid DNA, very low proliferation, and low RNA, similar to that found by use to be typical for normal B cells. In chronic myelogenous leukemia, two cell populations were distinguished, one with high RNA, the other with very low RNA and elongated nuclear diameter due to stripped, unfolded nuclei of polymorphonuclear leukocytes. The number of leukemic blast cells, identified by aneuploid DNA values, correlates well with conventional microscopy counts and could be followed during the course of treatment. Thus, acridine orange flow cytometry can be used to discriminate subtypes of human leukemias, to determine cell cycle stages, and to detect and monitor aneuploid leukemia stemlines.</jats:p

Discrimination of human leukemia subtypes by flow cytometric analysis of cellular DNA and RNA

A newly developed flow cytometry technique for simultaneous measurements of three features of individual cells--DNA, RNA, and nuclear diameter--using acridine orange as a fluorescent metachromatic dye, has been applied to cell-cycle analysis. DNA stemline determination, and to classification of 102 cases of human leukemias in adults. Acute lymphoblastic leukemia (L1–2) was characterized by moderately increased RNA of G0/1 cells as compared to normal lymphocytes; acute nonlymphoblastic leukemia (M 1–5) by very high RNA of G0/G1 cells. Both had either diploid or aneuploid DNA stemlines. Chronic lymphocytic leukemia showed diploid DNA, very low proliferation, and low RNA, similar to that found by use to be typical for normal B cells. In chronic myelogenous leukemia, two cell populations were distinguished, one with high RNA, the other with very low RNA and elongated nuclear diameter due to stripped, unfolded nuclei of polymorphonuclear leukocytes. The number of leukemic blast cells, identified by aneuploid DNA values, correlates well with conventional microscopy counts and could be followed during the course of treatment. Thus, acridine orange flow cytometry can be used to discriminate subtypes of human leukemias, to determine cell cycle stages, and to detect and monitor aneuploid leukemia stemlines.</jats:p

p15(Ink4b) is a critical tumour suppressor in the absence of p16(Ink4a)

The CDKN2b-CDKN2a locus on chromosome 9p21 in human (chromosome 4 in mouse) is frequently lost in cancer. The locus encodes three cell cycle inhibitory proteins: p15(INK4b) encoded by CDKN2b, p16(INK4a) encoded by CDKN2a and p14(ARF) (p19(Arf) in mice) encoded by an alternative reading frame of CDKN2a (ref. 1). Whereas the tumour suppressor functions for p16(INK4a) and p14(ARF) have been firmly established, the role of p15(INK4b) remains ambiguous. However, many 9p21 deletions also remove CDKN2b, so we hypothesized a synergistic effect of the combined deficiency for p15(INK4b), p14(ARF) and p16(INK4a). Here we report that mice deficient for all three open reading frames (Cdkn2ab(-/-)) are more tumour-prone and develop a wider spectrum of tumours than Cdkn2a mutant mice, with a preponderance of skin tumours and soft tissue sarcomas (for example, mesothelioma) frequently composed of mixed cell types and often showing biphasic differentiation. Cdkn2ab(-/-) mouse embryonic fibroblasts (MEFs) are substantially more sensitive to oncogenic transformation than Cdkn2a mutant MEFs. Under conditions of stress, p15(Ink4b) protein levels are significantly elevated in MEFs deficient for p16(Ink4a). Our data indicate that p15(Ink4b) can fulfil a critical backup function for p16(Ink4a) and provide an explanation for the frequent loss of the complete CDKN2b-CDKN2a locus in human tumours