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. 2003 Apr;23(7):2530-42.
doi: 10.1128/MCB.23.7.2530-2542.2003.

Dissecting the contribution of p16(INK4A) and the Rb family to the Ras transformed phenotype

Philip J Mitchell  1 , Elena Perez-Nadales, Denise S Malcolm, Alison C Lloyd
Affiliations

Dissecting the contribution of p16(INK4A) and the Rb family to the Ras transformed phenotype

Philip J Mitchell et al. Mol Cell Biol. 2003 Apr.

Abstract

Although oncogenic Ras commonly contributes to the development of cancer, in normal primary cells it induces cell cycle arrest rather than transformation. Here we analyze the additional genetic changes required for Ras to promote cell cycle progression rather than arrest. We show that loss of p53 is sufficient for oncogenic Ras to stimulate proliferation in the absence of extrinsic mitogens in attached cells. However, surprisingly, we find that p53 loss is not sufficient for Ras to overcome anchorage dependence or contact inhibition. In contrast, expression of simian virus 40 (SV40) large T antigen (LT) allows Ras to overcome these additional cell cycle controls. Mutational analysis of SV40 LT shows that this action of SV40 LT depends on its ability to inactivate the retinoblastoma (Rb) family of proteins, in concert with the loss of p53. Importantly, we show that inactivation of the Rb family of proteins can be mimicked by loss of the cyclin-dependent kinase inhibitor p16(INK4A). p16(INK4A) is commonly lost in human tumors, but its contribution to the transformed phenotype is unknown. We demonstrate here a role for p16(INK4A) in the loss of cell cycle controls required for tumorigenesis and show how accumulating genetic changes cooperate and contribute to the transformed phenotype.

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Figures

FIG. 1.
FIG. 1.
Loss of p53 function is sufficient for Ras induced proliferation in the absence of mitogens. (A) Phase-contrast images of pools of Schwann cells expressing either a dominant-negative mutant of p53 (dnp53) or the SV40 LT, together with HRasVal12 (Ras) or the empty retroviral vector LXSN. (B) Asynchronous populations of Schwann cells were cultured in normal culture medium or defined medium containing no mitogen for 40 h. The percentage of cells entering S phase was determined by measuring BrdU incorporation 12 h prior to fixing and staining. The error bars indicate the standard deviations of triplicate assays.
FIG. 2.
FIG. 2.
Loss of p53 is not sufficient for Ras to overcome contact inhibition or for anchorage-independent proliferation. (A) Percentage of Ras or LXSN-infected Schwann cells, expressing dnp53 or SV40 LT (LT), which proliferated to form foci in a confluent monolayer after 2 weeks, as visualized by staining with methylene blue. A representative focus of SV40 LT cells (LT) expressing Ras is shown. (B) Percentage of Schwann cells expressing dnp53 or LT, together with Ras or the empty vector LXSN, which proliferated to form colonies in soft agar. Pools of cells (4 × 103) were seeded into soft agar. After 2 weeks the colonies were photographed and counted. Representative colonies of cells expressing Ras, together with dnp53 or LT, are shown. Assays for panels A and B were carried out in duplicate and are representative of three independent experiments.
FIG. 3.
FIG. 3.
The N terminus of LT is responsible for abrogating the additional checkpoints that enables Ras to be proliferative in the absence of anchorage. Pools of drug selected Schwann cells (4 × 103), expressing dnp53 and the N terminus of LT together or on their own with Ras or the empty retroviral vector LXSN, were seeded in soft agar. After 2 weeks the colonies were stained with neutral red prior to being counted and photographed. Assays were carried out in duplicate, and a representative result of three independent experiments is shown.
FIG. 4.
FIG. 4.
Inactivation of both p53 and the Rb family are required for Ras-dependent, anchorage-independent proliferation. Pools of drug selected Schwann cells (4 × 103) expressing dnp53 with wild type (WT) or mutants of the N terminus of LT (N-LT), together with Ras or the empty vector LXSN, were seeded in agarose. After 2 weeks the colonies were stained with neutral red prior to being counted and photographed. Assays were carried out in duplicate, and a representative result of three independent assays is shown.
FIG. 5.
FIG. 5.
Spontaneous acquisition of anchorage-independent proliferation is associated with frequent loss of p16INK4A expression. (A) Colonies of cells expressing dnp53 and Ras that proliferated in soft agar were picked, dissociated, and expanded in culture. A total of 4 × 103 cells were reseeded in agarose. Two weeks later the colonies were photographed. The parental polyclonal dnp53/Ras-expressing cells are shown adjacent to three clones. Assays were carried out in duplicate, and a representative of three independent assays is shown. (B) Immunoblots of lysates prepared from 10 dnp53/Ras-expressing clones that had acquired the ability to proliferate efficiently in soft agar. p16INK4A mRNA levels were determined by semiquantitative RT-PCR.
FIG. 6.
FIG. 6.
p16INK4A loss is responsible for conferring anchorage independence. (A) Pools of LTRas and anchorage-independent dnp53Ras Schwann clones that no longer expressed endogenous p16INK4A, expressing either wild-type rat p16INK4A or the empty vector pWZLhygro, were seeded in soft agar. The graph is representative of two independent experiments showing the average percentage of cells seeded that proliferated to form colonies in soft agar. The error bars represent the standard deviations of triplicate assays. The levels of p16INK4A as determined by immunoblotting are shown in the inset panel. (B) Populations of drug-selected Schwann cells, expressing dnp53 with the p16INK4A-insensitive mutant of cdk4 (cdk4R24C), antisense p16INK4A, or the N terminus of LT, together with Ras or the empty vector LXSN, were seeded in agarose. After 2 weeks the colonies were stained with neutral red prior to being counted and photographed.
FIG. 7.
FIG. 7.
Anchorage-independent Ras/dnp53 cells phosphorylate Rb in suspension and maintain Rb dependency for proliferation. (A) Antibody phospho-Rb (Ser-795) recognizes Rb in proliferating (b) but not in quiescent (a) rat Schwann cells. Cells from growing colonies in soft agar were removed and spun onto coverslips and immediately fixed. The cells were immunostained for either a cytoplasmic protein S100 (c) or phospho-Rb (d and e, which show examples of cells negative and positive for phospho-Rb staining, respectively). (B) BrdU incorporation of cells after transfection with a nonphosphorylatable form of Rb (12) or control vector. Four NSdnp53Ras clones are shown. Clones 6 and 7 are p16INK4A positive; clones 8 and 9 are p16INK4A negative. NSLT are Schwann cells expressing SV40 LT. Transfected cells were monitored by determining GFP expression. The error bars indicate the standard deviations of triplicate assays.
FIG. 8.
FIG. 8.
Contribution of the inactivation of checkpoints to Ras transformation. Model of how the progressive loss of checkpoints allows Ras to act positively on the cell cycle. Loss of p53 activity causes Ras to be proliferative in the absence of mitogens. Inactivation of the Rb family, by loss of p16INK4A or by inactivation by the N terminus of SV40 LT, renders the cells capable of proliferation independently of anchorage and insensitive to contact inhibition.
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