Transcriptional networks and cellular senescence in human mammary fibroblasts

Abstract

Senescence, the molecular program that limits the finite proliferative potential of a cell, acts as an important barrier to protect the body from cancer. Techniques for measuring transcriptome changes and for modulating their expression suggest that it may be possible to dissect the transcriptional networks underlying complex cellular processes. HMF3A cells are conditionally immortalized human mammary fibroblasts that can be induced to undergo coordinated senescence. Here, we used these cells in conjunction with microarrays, RNA interference, and in silico promoter analysis to promote the dissection of the transcriptional networks responsible for regulating cellular senescence. We first identified changes in the transcriptome when HMF3A cells undergo senescence and then compared them with those observed upon replicative senescence in primary human mammary fibroblasts. In addition to DUSP1 and known p53 and E2F targets, a number of genes such as PHLDA1, NR4A3, and a novel splice variant of STAC were implicated in senescence. Their role in senescence was then analyzed by RNA silencing followed by microarray analysis. In silico promoter analysis of all differential genes predicted that nuclear factor-kappaB and C/EBP transcription factors are activated upon senescence, and we confirmed this by electrophoretic mobility shift assay. The results suggest a putative signaling network for cellular senescence.

Figure 1.

Outline of the experimental conditions used for microarray comparisons. Primary breast fibroblasts (HMF3) were either immortalized with the catalytic subunit of human telomerase (hTERT) and wild-type LT antigen (LTwt), conditionally immortalized with hTERT and temperature-sensitive LT antigen (LTts), or passaged through to senescence. The HMF3Dwt cells have LTwt and continue to proliferate when cultured at 39°C. The HMF3A cells have LTts and become senescent after 7 d at 39°C. RNAi lines were created by introducing pSUPER vector constructs into HMF3A lines.

Figure 2.

Changes in protein and DNA binding levels upon LT inactivation and reactivation (→ 33). Westerns were used to measure protein levels (A). EMSA were used to measure N-myc, C/EBP, and NF-κB DNA binding activity (B). The average ratios for the densitometry readings are given in relation to the level in HMF3A cells at 33°C. Supershifts using C/EBPα and β antibodies indicate that both of these proteins are involved. Western blots also were performed with these antibodies. Cold competition (cc) with unlabeled probe shows the specificity of the EMSA bands. Westerns were performed at least twice using different lysates. EMSA were performed three times using different lysates.

Figure 3.

Differences in the RNAi lines. RT-PCR was used to determine the effectiveness of the stable RNAi vectors against BTG2, BTG3, NR4A3, and PHLDA1. (A) HMF3A cells containing the various p-SUPER vectors were shifted to 39°C for 7 d, and 3A-BTG2i cells also were shifted back to 33°C for 7 d. (B) The protein levels for MKP-1 (product of the DUSP1 gene) and IL-1β were examined for the various RNAi cultures after the cells were shifted to 39°C for 7 d. (C) The DNA binding activity of AP-1 was measured by EMSA after 7 d of LT inactivation in the DUSP1i and control cells. A cold competition (cc) control sample was used to assess the specificity of the band. (D) Staining of cells shifted to 39°C for 2 wk and then either shifted back to 33°C for 2 wk (back to 33°C) or left at 39°C for another 2 wk (39°C) was performed for 3A-STACβI, 3A-PHLDA1i, and control cells. (E) Comparison of the structure of STAC and the splice variant STACβ. RT-PCR were performed with various primer combinations (1–6). If STACβ has the same transcription start site as STAC, it would code for a shorter protein with the sequence given here, with the underlined area indicating where it would be different from STAC. For RNAi, a target sequence (+) was used that was only in STACβ. The resulting cells (39^*) did have less STACβ mRNA than control cells, when compared after shifting of both cells to 39°C for 7 d. RT-PCR were performed three times with different RNA lysates. In addition to this samples were taken at three different times to ensure that the reaction was in the linear amplification stage. Westerns were performed at least twice by using different lysates. EMSA were performed three times by using different lysates.