Chronology and pattern of human chromosome replication, IV. Autoradiographic studies of binucleate cells.

Abstract

The chronology and pattern of chromosomal DNA replication in mammalian cells, including human, have been studied almost exclusively in uninucleated cells. Except for some data recently obtained in our laboratory on human tetraploid cells,' we are unaware of reports dealing with the sequence of DNA duplication of chromosomes in binucleate cells. Asynchrony of chromosomal DNA replication in various diploid human cell lines has been demonstrated by a number of workers using autoradiographic techniques.2-'0 A recent publication suggests that all chromosomes appear to finish DNA replication at the same time, but approach that end at different rates, with one of the X chromosomes in the normal female replicating much more rapidy than the other chromosomes during the last few hours of the S-period. A similar pattern has been observed in our laboratory for tetraploid cells.1 iVIost important, almost all of the chromosomes of a given group of homologues of a tetraploid cell were labeled to some degree when the tritiated thymidine was pulsed for 10 inin, 4-6 hr before fixation. In these cells, marked labeling of all four homologues was evident when the isotope was in contact with cultured cells for 2 hr. The availability of a human diploid cell line with an impressive number (1-4%) of binucleate cells offered the unusual opportuniity of examining the chronology of chromosomal replication in the two nuclei. The study indicates that one nucleus replicated its DNA considerably ahead of the other, and then awaited completion of DNA replication in its mate before going into metaphase. Materials and Mlethods.-The cell line studied (RPMI #8205, MC #2) was derived from the blood of a 54-year-old womani with acute myeloblastic leukemia. The frequency of binucleate cells in the cultured line varied from 1.3% to nearly 4%. Cells were considered to be binucleate when surrounded by one cellular membrane and containing two distinct nuclei in a common cytoplasm. This cell line was cultutred originally in MIcCoy's medium with 20% calf serum and 0.2% human albumin. Subsequently, the cells used in the present studies were cultured in suspension in RPMI media,'2 usually in 16-oz bottles made by Brockway. Details regarding the cell line will appear elsewhere.'3 Tritiated thymidine (specific activity 6.7 c/mM) was added to the culture (final concentration of 1 ,uc/ml) for periods of time ranging from 20 min to 5 hr before fixation and left in the culture for the total interval of time unless pulse experiments are indicated. Most of the details regarding the processing of the cells in preparation for chromosome analysis and for autoradiography have been described in previous publications.9 '0 Colchicine (1 ,ug/ml of medium) was present in the culture medium during the last 2 hr of incubation. At the end of incubation the culture was transferred to centrifuge tubes, and the cells were packed by centrifugation. The supernatant was discarded and the cells were then washed several times with Hank's solution, followed by treatment with hypotonic solution in order to swell the cells prior to spreading of the chromosomes. The material was then fixed in acetic-alcohol (1: 3), and slides were prepared by an airdrying technique and stained with acetic orcein. These techniques cause little breakage and maintain cellular integrity as ascertained by the persistence of the cytoplasm and an intact cellular membrane surroundinig the metaphase chromosomes. Under these conditions the metaphase chromosomes of two contiguous cells can be easily differentiated by their morphological appear-