Replication synchrony-PCR: a sampling-time-independent assay for replication synchrony in human tissues and tumors in situ

Abstract

Replication synchrony within a cell population can be demonstrated by pulse-labeling followed by PCR amplification of immunoprecipitated 5-iodo-2'-deoxyuridine (IdUrd)-labeled DNA from cells of otherwise indeterminant kinetic stages. This replication synchrony-PCR approach may be valuable in understanding the dynamics of human normal tissue or solid tumor replication in situ where access for repeated sampling is severely limited. IdUrd labeling provides a sampling-time-independent method for assessing the replicative status of a cell population at the time when the label was presented. Using genes whose time of replication in S phase is already known, the presence of a cell in early or late S phase can be determined and a qualitative measure made of replication synchrony in the population. This approach was evaluated in synchronous and random cultures of Ej cells using the early replicating PGK-1 gene to identify cells in early S phase at the time of labeling and the late replicating factor IX gene to identify cells that were in late S phase. To test the feasibility of clinical application of this technique, human tumor cells from patients with advanced cancers, given IdUrd therapeutically at specified times of the day, were evaluated. In some patients, replication synchrony-PCR provided evidence of parasynchronous DNA replication in tumor cells. This technique could be appended to existing clinical studies in which BrdUrd or IdUrd is being given to patients either diagnostically or therapeutically.

Figure 1

Synchrony in human Ej cells. Three 75-cm² flasks of mitotically selected cells were labeled with IdUrd for 1 h at hourly intervals through the cell cycle, and the IdUrd-positive and bead-standardized fluorescence intensity were determined for each of 17 time points. The IdUrd-positive fraction was determined by gating from the bivariate histograms obtained from flow analysis of pulse labeled samples (□). A flask was reserved for digital time-lapse analysis and subsequently scored for the occurrence of mitoses (•).

Figure 2

Bivariate flow cytometry of synchronous samples used for replication timing analysis. Cells 5–6 h (a) and 14–15 h (b) after mitotic selection were used for PCR analysis along with randomly growing cells similarly pulsed (d). A flask pulsed at 4–5 h was chased for an additional 24 h before being fixed for flow analysis (c). Two-dimensional histograms of IdUrd immunofluorescence versus propidium iodide are shown for these samples.

Figure 3

Analysis of PCR products of immunoprecipitated DNA from synchronous Ej cultures. Immunoprecipitated DNA from Ej cultures pulsed for 1 h with IdUrd in early S phase (lane E) and late S phase (lane L) as well as randomly labeled cells (lane R) were subjected to PCR for PGK-1 (Left) and F9 (Right).

Figure 4

Bivariate flow cytometry of IdUrd-positive tumor cells. IdUrd positive and negative cells in tumor antigen cell positive washings from patient ML with ovarian cancer given IdUrd at a dose of 3,750 mg/m² are shown. Cells were collected and fixed 24 h after beginning treatment.

Figure 5

Analysis of PCR products of immunoprecipitated DNA from patient samples. Immunoprecipitated DNA from patient samples labeled at 11 a.m. (patient ML) and at 8 p.m. (patient BP) were subjected to PCR for PGK-1, with and without previous HpaII digestion (Left) and F9 (Right). Lanes marked STD are standards for the PCR reactions.