Historia en la relación de la delincuencia y las drogas

The lack of any radiation induced p53 protein in SV-HUC-1 cells was not unexpected due to the presence of the SV40 T-antigen and/or the mutant protein leading to the accumulation of high levels of non-functional p53. In addition there was an apparent corresponding absence of any radiation induced cell cycle delay, (neither Gi nor G2/M arrest) as measured by flow

cytometry in SV-HUC-1 cells. Although this agrees with the previously reported observations of Maltzman and Czyzyk (1984), cell lines containing the T-antigen due to immortalisation by the SV40 virus are often more radioresistant than the corresponding non-immortalised cells from which they were derived (Arlett et al, 1988). In view of the proposed function of wild type or non-oncoprotein bound p53, i.e. as a specific transcriptional protein regulating down stream events that controls regulation of the cell cycle in response to DNA damage, it is difficult to reason why such cell lines would have an increased radioresistance conferred to them.

The lack of any inducible p53 protein or cell cycle delay in T24 cells containing no viral sequences and normal levels of wt p53, may reflect differences between normal and cancer cell types even when mutant p53 protein is not involved. Any protein which binds to wt p53 so abolishing its normal function could achieve this goal. Such a protein has recently been found in the form of the MDM2 protein reported in human sarcomas by Oliner et al (1992). The examination of a greater number of such cell lines will provide more conclusive data.

The only cell line in this study which did show both a radiation induced p53 protein increase and a cell cycle delay was HPV. The increased level of wt p53 (approximately two fold) in HPV cells following irradiation was not as great as the 3-8 fold increase reported to occur at Gi arrest by Maltzman and Czyzyk (1984) and Kastan et al (1991). An increase in the G2/M

population and not Gl was observed with HPV cells following irradiation. 5.10. Possible controlling mechanisms of p53 and cell cycle delay.

It is possible that this G2/M delay is not directly caused by elevated p53 levels

but as a consequence of a control mechanism affecting a number of other factors which act to delay the cell cycle not only at Gl but also at G2 following

radiation induced DNA damage. Wt p53 protein may be expressed at an increased level, due to increased stabilization via phosphorylation events, interaction with other proteins or oligomerization (Lane and Crawford 1979, Finlay et al, 1998, Hupp et al, 1992). However, this protein must also be in an active form to achieve its regulatory function. A possible candidate for such a control mechanism are the changes in phosophorylation of p3 4^dc2

kinase which alters it's kinase activity, y irradiation of Chinese hamster ovary cells has been shown to cause a rapid inhibition of p3 4cdc2 kinase

I N D U C T I O N O F p 5 3 1 1 1

activity and subsequent G2 arrest (Lock and Ross, 1990). The wild type p53

protein is also affected by p3 4^dc2 kinase acivity, becoming more

phosphoryiated in S phase (Bischoff et al., 1992). Hence a reduction in p34 <^dc2 kinase activity in response to radiation damage would lead to a less phosphoryiated p53 protein molecule which in turn would inhibit progression of the cell into or through S-phase. Alternatively if such a pathway was no longer functioning then the same kinase also appears to have the ability to regulate cell cycling via a control mechanism acting at G2- (See Figures 5.13 and 5.14 for an overview of possible control mechanisms acting at Gi and G2 respectively). Finally it should be noted that this increase

in p53 protein is reported to occur through a post transcriptional mechanism since mRNA levels do not change significantly following irradiation (Kastan

et al., 1991). Also that the mechanism of control of this protein is probably

not a simple one since it is acted upon by other kinases/ phosphatases such as protein phophatase 2A (PP 2A) which dephosphorylates p53 (Scheidtman

et al, 1991).

5.11, To what extent can this model for p53 function be relied upon?

The overwhelming body of evidence now supports the theory of wt p53 as a tumour suppressor gene. The protein which is coded for by this gene, although not essential for development does appear to be needed to limit damage to the DNA and maintain tlie integrity of the genome. (Mice having no p53 genes develop normally but suffer from a high incidence of tumours in a variety of organs early in life, Donehower et al, 1992).

However, caution should be exercised when interpreting the data concerning DNA damage induced cell cycle delay, both in this study and others which have used flow cytometry to analyse such changes. Although

FIG. 5.13.

Gl

late group

p53 bound to DNA at C-terminal domain resulting in transcriptional modification of downstream genes (possibly DNA polymerase a) so leading to arrest/inhibition of DNA synthesis.

p34odc2

+ve _{Active/functional kinase activity of}