LOS PRINCIPALES TIPOS DE PUENTES DE HORMIGÓN

Since the CIP/KIP proteins are reputed to act as assembly factors for cyclin- CDK complexes in some circumstances (Cheng et al., 1999; LaBaer et a i, 1997; Parry et a i, 1999; Zhang et a i, 1994), the subunit rearrangement observed upon temperature shift in SVtsS cells would be readily explained by the restoration of p21^^^ expression. To explore this possibility, recombinant retroviruses were used to express ectopic p21^^^^ or p27^^^^ in SVtsS cells growing at the permissive temperature.

A)34°C

B) 38.5°C

Figure 3.10. Subcellular location of cyclin Dl in SVtsS cells cultured at the permissive and non-permissive temperatures.

SVtsS cells were seeded at the appropriate density at either 34°C (A) or 38.5°C (B) for 48 hours prior to fixing in 3% paraformaldehyde. Cyclin Dl was localised by staining with a polyclonal antibody (287.3) before viewing with a Zeiss laser scanning microscope. Left panels: 63x magnification, right panels: 5- fold enlargement of left panels.

To permit uptake of ecotropic retroviruses, SVtsS cells were infected with an amphotropic retrovirus (pWXL-neo-Eco) encoding the mouse basic amino acid transporter, which serves as the cell surface receptor for mouse ecotropic retroviruses (McConnell et al., 1998; Serrano, 1997). The amphotropic retrovirus (pWXL-neo-Eco) was produced from AM-12 cells stably expressing the retrovirus and the supernatant from these cells was added directly to growing SVtsS cells. Infected SVtsS cells were selected in 300 pg/ml geneticin for two weeks and pools of resistant cells (named SVtsS-ER^^J were used for subsequent infections with pBabe/7MTO ecotropic retroviruses.

Infectious viruses, encoding empty vector (pBabepwm), p21^^^ or p 2 7 ^ \ were produced by transient transfection of the BOSC-23 packaging cell line, and the supernatants were added directly to the SVtsS-ER^g^ cells. The retroviral vector conferred resistance to puromycin, enabling the rapid selection of pools of infected cells. Infection efficiencies were approximately 50% as judged by the percentage of cells surviving in puromycin.

In order to determine the relative expression levels of p 2 l‘^°’* and p 2 7 ^ \ cell lysates were prepared 6 and 12 days post-infection and analysed by direct immunoblotting with the respective antisera (Figure 3.11.). By six days post­ infection, the cells were expressing substantial levels of either p21^^^ or p 2 7 ^ \ In the case of p21^^% the ectopic expression levels were comparable to those of endogenous p21^^ in SVtsS cells at the non-permissive temperature (Figure 3.11.). With p27*“ ’\ endogenous levels were extremely low and gave no appreciable signal under the conditions used.

It was of interest to see whether the ectopic expression of p21^°"^ or p27™** had an effect on cellular proliferation of SVtsS cells at the permissive temperature. At 6 days post-infection, 2.5x10^ cells expressing empty vector, p21^^^ or p27*^* were seeded in 24 well plates and their proliferation followed for 6 days. The assay measured the uptake of crystal violet into viable cells (Materials and Methods) and was validated by confirming the linear relationship between absorbance of eluted stain (Ajçonm) and cell number (Figure 3.12.A). Both p21^^^ and p27^^^^ had a pronounced effect on cell proliferation, and by 12 days post-infection, cell numbers

SVts8-ER vector control recombinant virus 12 12 D a y 34 38.5 34 34 34 34 Temperature ("C)

Figure 3.11. Ectopic expression of CIP/KIP proteins in SVtsS cells cultured at the permissive temperature.

SVts8-ER cells were infected with recombinant ecotropic retroviruses encoding p21^^^^ and p27*^^^ and selected in 2.5 pg/ml puromycin. Six and twelve days after infection, lysates were prepared in NP40 lysis buffer and samples (30 pg) of protein were separated by SDS-PAGE in a 12% acrylamide gel and immunoblotted with antibodies against p21^^^^ (sc-397) and p27^^^ (sc-528). Endogenous p21^^^^ and p27^^^ from SVts8-ER cells at 34°C and 38.5°C were also analysed.

A) 2.5 2.0 cell number -2 o (A 0.5 0 200000 400000 600000 cell number B) 125 100

I

Figure 3.12. Growth arrest of SVtsS cells at 34°C following ectopic expression of p21^***'and p27'^****

A) SVts8-ER cells were seeded into 24 well plates at increasing densities. For each specified cell number, cells were fixed in 10% formaldehyde and viable cells stained with crystal violet. The eluted stain was determined by measurements of A5 9 0nm- A Calibration curve was plotted of against increasing cell number. B)

SVts8-ER cells were infected with pBabepwm viruses encoding p21^^^ or p27^^^ and selected in 2.5 pg/ml puromycin for 6 days. After selection, cells were transferred to 24 well plates (2.5x10^ cells/well) and followed for 6 days. For each time point cells were treated as in A. Values were normalised against cell number at day one. Values were determined in triplicate and error bars represent standard deviations.

were less than 20% of the vector only controls (Figure 3.12.B). Since these were pools of infected cells, there was likely to be some variation in expression levels and not all the cells were arrested.

A similar partial effect was observed in a BrdU incorporation assay (Figure 3.13.). BrdU is an analogue of thymidine and it can be incorporated in the DNA of cells as they go through S phase. The percentage of cells incorporating BrdU in a given time period gives an indication of the proliferative state of that cell population. For this assay, sub-confluent cells were labelled with 10 |iM BrdU for 4 hours, at 6 days after infection, i.e. immediately prior to plating for the proliferation assay. 27.5% of the cells in the pool infected with vector alone (pBabepwm) incorporated BrdU compared to only 11% of the cells infected with p21^^' and 8% of the cells infected with p27“ ^‘ (Figure 3.13.). As judged by SA-P-galactosidase expression (not shown), a proportion of the infected cells arrested with a senescence-like phenotype reminiscent of SVtsS cells at the non-permissive temperature.

3.6. Reassembly of cyclin Dl-CDK complexes by ectopic expression of CIP/KIP