Discussion

Cyclin D l is not a transcriptional target of Myc b u t Myc m ay well interact w ith the cyclin D l prom oter. O ur results show that the MycER p ro tein activated by OE is capable of trans-activating cyclin D l. This induction has been reported by Daksis et w ith very similar results. In addition, these authors show that the effect is transcriptional and that

Figure 5.9 GV-MycS cells induce Myc and enter the cell cycle

A FACS analysis

FACS analysis of cells grown to confluence, serum starved for 48 hours and treated for 24 hours w ith either 10% serum , OHT or left untreated. Shown is the proportion of cells plotted against DNA content.

B G raphical representation

Shown is the proportion of cells in the S and G2 phases of the cell cycle following each treatm ent fom p art A above.

C Myc induction

Cells w ere either (left two lanes) growing exponentially in 10% serum and treated w ith EtOH or OHT for 2 hours or (right six lanes) grow n to confluence, serum starved 48 hours and treated w ith OHT for the indicated time. They w ere then harvested and RNA p rep ared by the 'acid phenoT m ethod and analysed by n o rth ern blot. Show n is the hybridisation to the hum an c-Myc fragment from BJ3-Myc.

S + C2 200 400 Propidium iodide 600 350 H

Serum

Propidium iodide EtOH Serum OHT

OHT

4 * i l

Figure 5.10 Cyclin D l is not induced in Rat-1 GV-Myc 5 cells

Shown is a quantification using a phosphor-im ager of the northern blot in Fig. 5.9, probed for Cyclin D l, Myc and GAPDH. Cells w ere either (left tw o pairs of columns) grow ing exponentially in 1 0% serum and treated w ith EtOH or OHT for 2 hours or (right six pairs of columns) grow n to confluence, serum starved 48 hours and treated w ith OHT for the indicated time. Shown are the relative expression levels of cyclin D l an d Myc norm alised to the level of GAPDH, com pared w ith the uninduced state.

.2

10% serum 0% serum, stimulated with OHT for the indicated time in hours

protein levels also increase. MycER w hen activated by OHT it is not capable of inducing cyclin D l expression. Furtherm ore, the Myc trans­ activation dom ain (amino acids 106-143) is required for O E-dependent trans-activation. The effect is specific to cyclin D l and we know th at

MycER can trans-activate from CACGTG (and

also chapter 3) and that the cyclin D l prom oter contains such sites, v/hich suggests that the Myc DNA binding dom ain may also be required although I have not tested this. The effect is also dependent on the Myc N -term inus. A likely explanation therefore is that the Myc portion of MycER recognises the cyclin D l prom oter, while the Myc transactivation dom ain acts in concert w ith the activated Taf-II dom ain of the oestrogen receptor to stimulate transcription.

One of the reasons I initially considered cyclin D l as a Myc target was data on signal transduction by the CSF-1 receptor (CSF-IR). In NIH 3T3 cells, CSF-1 R activation leads to tyrosine kinase activity, binding to PI-3 kinase and induction of Fos, JunB and Myc. Cells expressing a CSF- IR m utant (tyrosine 809 to phenylalanine)^^^ fail to induce Myc b u t are norm al in the other responses to CSF-1 listed above. These cells fail to grow in semi-solid m edium , cannot grow in the absence of serum and do not induce cyclin D l. Enforced Myc expression restored the ability to grow^^^ and studies in macrophages^^^ indicate that cyclin D l induction is dow nstream of CSF-IR activation and of Myc. H ow ever, it has subsequently become clear th at the CACGTG sites in the cyclin D l prom oter w hich are conserved in mouse and are not in the serum -responsive part of the prom oter and this region in fact responds to Jun rather than Myc^^^^^"^.

A n interesting observation is that the basal level of cyclin D l ex p ressio n is g reatly red u c ed in MycER ex p ressin g cells. This phenom enon has been further studied by Eilers's group^^^ w ho show ed

th at Myc expression (both w ild type and MycER) represses cyclin D1 transcription. Interestingly, the effect does not require Myc Box II (amino acids 128-143) or the leucine zipper of Myc, and is independent of OHT addition in MycER cells, suggesting th at dim érisation w ith Max and DNA binding are not in fact required. It seems instead to operate by Myc competing for TFII-I w ith core prom oter bound USE. Thus, some effects of Myc on transcription m ay not operate via classical DN A-binding and trans-activation, but rather via the form ation of protein complexes. The evidence for this is that Myc can heterodim erise w ith both TFII-I^^^ and YY-I^^^ which are capable of binding to and activating transcription from the A d en o v iru s m ajor late p r o m o t e r ' ^ a s w ell as TA TA -less promoters"^^^ and the A deno-associated virus type 2 P5 promoter'^ respectively, through the initiator (Inr) sequences in each case. Myc prevents activation by USE (probably as a dim er w ith TFII-I) of the A denovirus major late prom oter Inr and it has been show n in an in vitro

system that Myc represses this prom oter by interfering w ith the TFII- I/T F II-D com plex. Since Myc in teracts w ith TFII-I, the sim plest explanation is a competition for this factor.

One m odel for the activation of cyclin D1 by MycER is that the MycER protein is tethered to the cyclin D1 prom oter by interaction w ith another protein (such as YY-I). Follow ing OF stim ulation, the Taf-II dom ain trans-activates transiently before Myc is complexed to Max, is rem oved from the cyclin D1 prom oter and binds to its ow n targets. W hether either the repression of cyclin D1 by Myc or its presence on the cyclin D1 prom oter is relevant to Myc function is still unclear.

That Myc, w hich is know n to prom ote cell cycle progression, sh o u ld repress a cyclin seem s contradictory. H ow ever, cyclin D1 expression does not correlate exactly w ith proliferation. A lthough it is induced by grow th factors 384-386^ it is also induced at senescence^^°

(when Myc is dow n regulated) and is induced in response to the tum our repressor, W hat is m ore, cyclin D1 overexpression can be antiproliferative, for example it is very difficult to obtain cyclin D1 over­ expressing clones'^^. Cyclin D1 expression is slightly different in various cell types b u t in rat fibroblasts, peaks following serum stim ulation and then decreases as cells cross the G l/S boundary. This dow n-regulation a n d /o r exclusion of the p ro tein from the nucleus is p ro b ab ly a requirem ent for progress th ro u g h the cell and is a possible p o in t of Myc action. It is also possible th at in Myc over- expressing cells, selection acts in favour of those w ith reduced levels of cyclin D1 as constitutive expression of Myc results in decreased levels of cyclin D l. In NIH 3T3 cells in contrast, Myc induces cyclin D1 translation via u p regulation of eIF-4E and repression is not observed*^^^. M arhin et

find that Myc alone is not sufficient to repress cyclin D l b u t that in conjunction w ith the loss of Rb, cyclin D l expression and kinase activity are rep ressed . D espite these fin d in g s, cyclin D l does p ro m o te proliferation in m any circum stances. D -type cyclins can rescue cells arrested by Rb overexpression^^^''^^^''^^^ and can shorten Cyclin D l m ay also be required for G1 to S progression since m icroinjection of cyclin D l antibodies or antisense arrests cells in Gl^^^. Cyclin D l is also linked to cancer; for exam ple it is found to be overexpressed in p a ra th y ro id adenomas'^^®, B-cell lymphomas'^^^, sq u a m o u s cell carcinomas^^*^, breast and oesophageal cancers (see H all and Peters, 1996431 Qj. M otokura and Arnold, 1993"^^^ for review). A lthough I failed to detect cooperation betw een activated Ras and cyclin D l in a REF transform ation assay (not shown), such a result has been reported by Lovec et the resu ltin g cells b ein g im m ortalised, anchorage independent and able to form fibrosarcomas in nude mice.

A better understanding of the relationship betw een cyclin D l and Myc will have to await delineation of the precise role for cyclin D l in the cell cycle; it is clear now that it acts as a complex w ith cdk-4 or cdk-6 and is able to phosphorylate Rb (review ed in Peters, 199#^^ and Sherr, 1994433) the G1 phase of the cycle. The o u tstan d in g question is w hether cyclin D l is required for M yc-dependent proliferation. The same techniques that dem onstrated that cyclin D l is required for G l/S progression (antisense and antibody injection) in norm al fibroblasts^^® could be applied to MycER cells to address this.