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. 2012 Mar 1;11(5):1008-13.
doi: 10.4161/cc.11.5.19492. Epub 2012 Mar 1.

The specific role of pRb in p16 (INK4A) -mediated arrest of normal and malignant human breast cells

Alexey V Bazarov  1 , Won Jae Lee, Irina Bazarov, Moses Bosire, William C Hines, Basha Stankovich, Agustin Chicas, Scott W Lowe, Paul Yaswen
Affiliations

The specific role of pRb in p16 (INK4A) -mediated arrest of normal and malignant human breast cells

Alexey V Bazarov et al. Cell Cycle. .

Abstract

RB family proteins pRb, p107 and p130 have similar structures and overlapping functions, enabling cell cycle arrest and cellular senescence. pRb, but not p107 or p130, is frequently mutated in human malignancies. In human fibroblasts acutely exposed to oncogenic ras, pRb has a specific role in suppressing DNA replication, and p107 or p130 cannot compensate for the loss of this function; however, a second p53/p21-dependent checkpoint prevents escape from growth arrest. This model of oncogene-induced senescence requires the additional loss of p53/p21 to explain selection for preferential loss of pRb function in human malignancies. We asked whether similar rules apply to the role of pRb in growth arrest of human epithelial cells, the source of most cancers. In two malignant human breast cancer cell lines, we found that individual RB family proteins were sufficient for the establishment of p16-initiated senescence, and that growth arrest in G 1 was not dependent on the presence of functional pRb or p53. However, senescence induction by endogenous p16 was delayed in primary normal human mammary epithelial cells with reduced pRb but not with reduced p107 or p130. Thus, under these circumstances, despite the presence of functional p53, p107 and p130 were unable to completely compensate for pRb in mediating senescence induction. We propose that early inactivation of pRb in pre-malignant breast cells can, by itself, extend proliferative lifespan, allowing acquisition of additional changes necessary for malignant transformation.

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Figures

Figure 1
Figure 1
Individual RB family proteins can compensate for each other in mediating p16-initiated growth arrest of MDA-MB-231 human breast cancer cells. (A) MDA-MB-231-TETp16 cells stably transduced with retroviruses encoding shRNAs against indicated RB family proteins were harvested and analyzed by immunoblotting. Prominent Ponceau S stained bands were used as loading controls. (B) Cells transduced with indicated shRNAs or vector alone were treated with 1 µM doxycycline for 48 h then harvested and subjected to propidium iodide (PI) staining and flow cytometry. Exponentially growing cells transduced with an empty vector were used as controls. (C) Cells transduced with indicated shRNAs or vector alone were treated with 1 µM doxycycline for 48 h then dissociated, counted and re-plated at clonal densities in the absence of doxycycline. After 3 weeks, the resulting colonies were counted and plating efficiency was calculated as (#colonies formed/#cells plated) × 100%. Relative plating efficiencies were then calculated by comparison to un-induced controls. Mean values and standard deviations (n = 3) are shown. (D) MDA-MB-231-TETp16 cells expressing HPV E6 or HPV E7 were either left untreated (-DOX) or treated with 1 µM doxycycline for 48 h (+DOX). The cells were then harvested, subjected to PI staining and analyzed by flow cytometry.
Figure 2
Figure 2
Suppression of pRb, but not p107 or p130, extends the proliferative lifespan of primary human mammary epithelial cells. (A) The relative expression of mRNAs encoding pRb, p107 and p130 was measured by qRT-PCR for actively growing HMECs stably transduced with retroviruses encoding shRNAs against the indicated proteins. Quantitative results were normalized to those of a stably expressed reference transcript encoding TATA binding protein (TBP). Mean values and SD (n = 3) are shown. (B) Total population doublings of HMECs stably transduced with retroviruses encoding shRNAs against the indicated proteins are plotted vs. time. (C) Representative morphologies of HMEC cultures expressing the indicated shRNAs (200x). The micrographs were taken 30 d (Vector, shp107, shp130), 52 d (shpRb), 42 d (shpRb/107/130) or 23 d (shp16) following viral transduction. (D) Total levels of cyclin E, p21 and p16 proteins were compared by immunoblotting in HMECs transduced with indicated shRNAs. Actin abundance was used as an indication of loading equivalence. The results shown are representative of results obtained using HMECs from two independent specimens. See additional results in Figure S2.
Figure 3
Figure 3
Cells expressing shRNAs against pRb were slightly less sensitive than cells expressing shRNAs against p107 or p130 to growth inhibition by a specific CDK inhibitor. The bar graph indicates PD 0332991 mean IC50 values (nM) for MDA-MB-231-derived cell lines transduced with shRNAs against indicated RB-family proteins. Standard deviations (n = 3) along with p values (Student t-test) are shown.
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