Alterations in replication timing of cancer-related genes in malignant human breast cancer cells

Abstract

The replication timing of nine genes commonly involved in cancer was investigated in the MCF10 cell lines for human breast cancer progression. Six of these nine genes are part of a constellation of tumor suppressor genes that play a major role in familial human breast cancer (TP53, ATM, PTEN, CHK2, BRCA1, and BRCA2). Three other genes are involved in a large number of human cancers including breast as either tumor suppressors (RB1 and RAD51) or as an oncogene (cMYC). Five of these nine genes (TP53, RAD51, ATM, PTEN, and cMYC) show significant differences (P < 0.05) in replication timing between MCF10A normal human breast cells and the corresponding malignant MCF10CA1a cells. These differences are specific to the malignant state of the MCF10CA1a cells since there were no significant differences in the replication timing of these genes between normal MCF10A cells and the non-malignant cancer MCF10AT1 cells. Microarray analysis further demonstrated that three of these five genes (TP53, RAD51, and cMYC) showed significant changes in gene expression (≥2-fold) between normal and malignant cells. Our findings demonstrate an alteration in the replication timing of a small subset of cancer-related genes in malignant breast cancer cells. These alterations partially correlate with the major transcriptional changes characteristic of the malignant state in these cells.

Fig.1. Singlet/Doublet Images

Representative FISH images of doublets (left row), singlet/ doublets (center row), and singlets (right row) are shown in BrdU positive cells (Blue = DAPI, red = BrdU, green = FISH signal) for the PTEN (A–C), PPF1A2 (D–F) and p53 (G–I) genes. Insets show FISH signals at higher magnification. Bars denotes 1 µM.

Fig. 2. Singlet doublet percentages for breast cancer related genes

The distribution of cells in S phase are shown for each gene that have both alleles replicated (doublets, white), only one allele replicated (singlet/doublet, dark gray) and both alleles not replicated (singlets, light gray). Nine of these genes (TP53, RAD51, PTEN, cMYC, ATM, RB1, CHK2, BRCA1, and BRCA2) are known to be involved in breast cancer progression (A–I). Two of these genes (ANO1 and TCN1) were chosen from a microarray from among those genes which changed the most in expression from 10A to CA1a (J–K). PPFIA2 is a known late replicating gene (L) and COL1A1 is a highly expressed gene in fibroblasts that is compared between WI38 fibroblasts and the MCF10 lines (M). The number of determinants for each gene ranged from 140 to 300. Error bars represent SEM.

Fig. 3. Cell cycle characterization of MCF10A lines

(A) Percentages of BrdU+/ BrdU− based on 300 cells ± SEM for each cell line. (B) Growth curves for each cell line. Average of 3 determinations ± SEM

Fig. 4. Average Replication Timing of Breast Cancer Related Genes

The average replication timing in hours was calculated based on a 10 hour S phase. The percentage of doublets + singlets/doublets was calculated for each gene shown in Figure 2, multipled by 10 and subtracted from 10 hours to estimate the average replication timing in hours. Based on the Chi squared test, 7 of 13 genes showed significant differences between 10A and 10CA1a (thick brackets), while only BRCA2 (thin bracket) was significantly different between 10A and 10AT1, and COL1A1 (dotted bracket) is statistically significant comparing MCF10 to WI38; *p<.05, **p<.01, ***p<.001.