Pretreatment with transforming growth factor beta-3 protects small intestinal stem cells against radiation damage in vivo

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1 British Jouml of Cncer (1997) 75(1), Cncer Reserch Cmpign Pretretment with trnsforming growth fctor bet-3 protects smll intestinl stem cells ginst rdition dmge in vivo CS Potten1, D Booth1 nd JD Hley2 'Pterson Institute for Cncer Reserch, Christie Hospitl NHS Trust, Wilmslow Rod, Mnchester M2 9BX, UK; 2Oncogene Science, 16 Chrles Lindbergh Blvd, Uniondle, New York , USA Summry The gstrointestinl trct, with its rpid cell replcement, is sensitive to cytotoxic dmge nd cn be site of dose-limiting toxicity in cncer therpy. Here, we hve investigted the use of one growth modultor to mnipulte the cell cycle sttus of gstrointestinl stem cells before cytotoxic exposure to minimize dmge to this norml tissue. Trnsforming growth fctor bet-3 (TGF-,3), known inhibitor of cell cycle progression through G,, ws used to lter intestinl crypt stem cell sensitivity before Gy of gmm irrdition, which ws used s model cytotoxic gent. Using crypt microcolony ssy s mesure of functionl competence of gstrointestinl stem cells, it ws shown tht the dministrtion of TGF-P3 over 24-h period before irrdition incresed the number of surviving crypts by four- to six-fold. To test whether chnges in crypt survivl re reflected in the well-being of the niml, survivl time nlyses were performed. After 14.5 Gy of rdition, only 35% of the nimls survived within period of bout 12 dys, while prior tretment with TGF-,B3 provided significnt protection ginst this erly gstrointestinl niml deth, with 95% of the treted nimls surviving for greter thn 3 dys. Keywords: trnsforming growth fctor bet; intestinl stem cells; cell cycle rrest; rdition protection; clonogenic cells Dmge to the stem cells of renewing tissues is serious dverse side-effect of cncer therpy tretments. The moist strtified epitheli (orl nd genitl) nd the simple columnr epitheli in the smll bowel represent the most sensitive of these renewing tissues. Dmge to the stem cells in these sites results in cellulr depletion which is mnifested t times to be equivlent to the trnsit time through the tissue. In murine smll bowel, this is equivlent to 3-4 dys nd in humn orl mucos to bout 1 dys. We designed series of experiments to investigte whether trnsforming growth fctor bet-3 (TGF-P3) could be used to modulte the sensitivity of gstrointestinl stem cells. With the incresing use of hemtopoietic cytokines to combt myelosuppression (Morstyn et l, 1994), gstrointestinl nd orl mucositis re incresingly becoming dose- nd schedule-limiting toxicities in stndrd- nd high-dose chemotherpy regimens. Chemotherpy gents tht cuse orl or gstrointestinl mucositis include commonly prescribed gents, such s 5-fluorourcil (5-FU), doxorubicin, vinblstine, methotrexte, txol nd etoposide. The incidence of gstrointestinl mucositis is thought to be similr to tht of orl mucositis, which occurs in pproximtely 2-25% of chemotherpy ptients (Chbner, 1993). The generl principles of such experiments fll within the outlines given below. An pproprite delivery of fctors to reversibly prevent cycle progression nd cuse n ccumultion of cells in G1 or Go before exposure to cytotoxic gent my render the stem cells more resistnt. In this wy, more stem cells might Received 3 September 1996 Revised 22 November 1996 Accepted 29 November 1996 Correspondence to: CS Potten survive the tretment nd be present to initite the regenertive process. An lterntive, or complementry, pproch might be to pply stimultory fctors tht shorten the cell cycle following exposure to cytotoxic gent, on the principle tht this might speed up the regenertive process. Clerly, combintions of inhibitors before cytotoxic exposure nd stimultors fter cytotoxic exposure might in theory represent the most effective pproch. It is interesting to note tht, if the principles of the experiment re vlid, reversl of the sequence, i.e. stimultors before or inhibitors fter cytotoxic exposure, might be expected to result not in protection but sensitiztion of the system. TGF-js hve been shown to reversibly inhibit the prolifertion of epithelil nd hemtopoietic progenitor cells in mid-lte G, phse (Polyk et l, 1994; Sherr nd Roberts, 1995). Mximl inhibition of cell cycling in unsynchronized cells requires tht TGF-P be present for one cell cycle period (Geng nd Weinberg, 1993). In the prevention of chemotherpy- or rdiotherpy-induced orl mucositis, it is criticl to optimize the length of time of TGF-P3 exposure, given the evidence tht TGF-f3 cts to protect cells by reversible inhibition of cell growth (Sonis et l, 1994). Sufficient preincubtion with TGF-P3 is required to tke cells out of cycle both before nd during exposure to chemotherpy while, idelly, entry of cells bck into cycle should occur s soon s the level of chemotherpeutic drug drops below toxic concentrtion. We report here the results of experiments using TGF-P3 dministered before rnge of doses of gmm rys on the survivl of intestinl stem cells. Stem cell function hs been mesured using the crypt microcolony regenertion ssy (Withers nd Elkind, 197; Potten nd Hendry, 1985, 1995) over rnge of gmm-ry doses nd by studying the survivl time of nimls receiving high doses of rdition. The TGF-P3 ws dministered over protrcted period of 24 h before the rdition exposure, nd this 1454

2 TGF-/33 protection in gut 1455 tretment fforded very significnt protection both within the microcolony ssy nd on whole niml survivl. The principles of the experimentl pproch, outlined bove, were tested by reversing the sequence (i.e. TGF-,B3 fter irrdition) in which cse sensitiztion ws observed. MATERIALS AND METHODS Animl studies BDF1 mice (Pterson Institute), ged 1-12 weeks nd weighing bout 25 g, housed under conventionl conditions with food nd wter d libitum nd 12-h light cycle (lights on t 7 h) were used throughout the experiment. For the crypt microcolony ssy, groups of six mice were treted t ech dose. Control nimls received either rdition lone or sline with.1% bovine serum lbumin (BSA) (the vehicle for TGF-P3). For the niml survivl time studies, groups of 2 mice were used for ech tretment protocol. All experimentl procedures were conducted in ccordnce with the recommendtions of the Animls (Scientific Procedures) Act 1986, UK. Irrdition methods For the crypt microcolony ssy, nimls were irrdited in cesium-137 gmm irrditor t dose rte of 3.5 Gry (Gy) min-1 delivered whole-body to nimls brething pumped ir. For the whole niml survivl studies, nesthetized nimls were irrdited with X-rys t 3 kvp (HVL 2.3 mm Cu) with the thorx, forelegs nd hed shielded. The dose rte ws.62 Gy min-'. We hve shown tht the crypt microcolony survivl curves generted using the cesium whole-body irrdition, nesthetized prtil-body X-rys nd whole-body X-rys nesthetized or unnesthetized were ll essentilly indistinguishble (Potten, unpublished dt). The pek in stem cell DNA synthetic ctivity in the smll intestine is observed t 3. h (Potten et l, 1977). Rdition doses were ll delivered t the sme time of dy (3. h; mking use of room where the light cycle ws reversed so tht the rdition could be delivered t 15. h rel time). The nimls were cclimtized to the reverse-cycle room for 2 weeks before n experiment. Production, isoltion nd use of recombinnt TGF-,3 Recombinnt TGF-P3 ws prepred either by purifiction of conditioned medi from CMV/TGF-,B3-trnsfected Chinese hmster ovry (CHO) cells (Stewrt et l, 1996) or by the refolding nd dimeriztion of TGF-P3 monomer expressed in E. coli, ccording to the method of Schlunegger et l (1992). Both TGF-P3 preprtions demonstrted the sme specific ctivity in vitro (IC5 35 pg ml-'; dt not shown) using stndrd CCL64 growth inhibition ssy (Iwt et l, 1985). The TGF-3 ws dissolved in sline with.1% BSA nd ws delivered intrperitonelly (i.p.) t doses between.5 nd 1,ug per mouse, per injection, with the mjority of the experiments using dose of 2.5 gg per mouse. A dose of 2.5 jg per mouse is equivlent on weight bsis to 1 jg kg-'. The injection protocols generlly involved four injections t 24 h, 8 h, 4 h before exposure to ionizing rdition nd h with being immeditely fter irrdition. This represented the stndrd delivery protocol. Vrious lterntive injection protocols were lso investigted Cncer Reserch Cmpign 1997 (-24, -16, -8, -4 nd h), rnging through to protocols in which TGF-,3 ws delivered following dose of rdition. Specificlly, other protocol tests were: -32, -24, -8, -4, ; -24, -8, -4,, +4; nd +8, +16, +24, +32 h, where + indictes the time fter irrdition nd - indictes the time before irrdition. Crypt microcolony ssy The crypt microcolony ssy (Withers nd Elkind, 197) is generlly ccepted s being test of the functionl cpcity of intestinl stem cells to regenerte nd mintin the intestinl epithelium. It hs been described extensively nd used widely (Potten et l, 1983). Briefly, it involves fixtion of the entire intestine 3-4 dys fter rdition nd the preprtion of routine 3- to 5-jim prffin sections stined with hemtoxylin nd eosin, cut trnsversely to the long xis of the intestine. This will generte cross-sections of the intestine, the perimeter or circumference of ech cross-section representing unit of length within which the number of crypts cn be counted. Ech crypt is closed prolifertive unit mintined by number of stem cells tht possess the bility to regenerte the crypt by process of clonl growth nd hence hve been termed clonogenic cells. A crypt cnnot be repopulted by cells from outside the crypt. Ech experimentl group contined six mice. From ech mouse ten cross-sections were obtined nd the number of regenerting crypts ws counted 3-4 dys fter irrdition. Thus, for ech experimentl group, 6 cross-sections were scored. As the probbility of observing regenerting crypt in section is dependent on the size of the structure, nd this my vry between different tretment groups, the size of 15 representtive crypts ws mesured t their widest point nd size correction ws pplied to the dt (Potten et l, 1981). For severl of the experiments, rnge of irrdition doses ws studied nd hence survivl curves for crypts could be generted. Dt for individul mice re presented on the grphs. The DR FIT progrm (Roberts, 199) ws used with multitrget model for fitting lines to such curves, for determining the prmeters tht define the curves with their confidence intervls, nd for tests of sttisticl difference between individul curves using vrince-rtio F-tests. A significnce level of.5 ws used. Survivl time experiments Groups of 2 mice were prtil-body irrdited with TGF-f3 dministrtion or sline/bsa before the irrdition ccording to the stndrd protocol. The mice were exmined four times dy for their helth sttus; ny moribund nimls were scrificed nd ny deths recorded. RESULTS Using five different rdition doses between 12 nd 16 Gy, the number of surviving crypts decresed exponentilly with incresing dose, s shown by Figure 1. There ws no sttisticlly significnt difference between the survivl curve obtined with rdition lone nd tht generted by prior injections of sline/bsa. The Do vlue defining the rdition lone curve is ± 17.2 cgy nd the extrpoltion number N is 3115 ± The Do is mesure of rdiosensitivity nd is the reciprocl of the slope of the exponentil prt of the curve. For the sline/bsa groups, the corresponding figures re Do = 112 ± 8.1 cgy nd N = 593 ± British Journl of Cncer (1997) 75(1),

3 1456 CS Potten et l c.1 - U, g.1 - ) C.. o.1-. Cu ) Q.o. * \' * Figure 1 Intestinl crypt survivl curves for nimls irrdited with no other mnipultions () nd nimls irrdited with series of pre-irrdition injections of sline/bsa t -24, -16, -8, -4 nd h (). Ech point represents the dt from n individul mouse nd there were six mice t ech dose for ech tretment protocol. Lines hve been fitted using the Puck formultion in the DR FIT progrm (Roberts, 199). No significnt difference between the two sets of dt ws observed (P =.1783) Figure 2 Intestinl crypts survivl curves for nimls pretreted with sline/bsa (-) with five injections (protocol ccording to Figure 1) or pretreted with four injections of 2.5 9g of TGF-f3 (U) t -24, -8, -4 nd h. A significnt difference between the two survivl curves ws observed (P <.1) As shown in Figure 2, four injections of TGF-,3 dministered t -24, -8, -4 h nd immeditely prior to dose of rdition results in survivl curve tht shows highly significnt shift (P <.1) to the right when compred with the curve for sline pretretment, indictive of incresed resistnce, i.e. protection of the clonogenic stem cells by the prior tretment with TGF-3. The prmeters tht define the TGF-P3 curves re Do = ± 1.6 cgy nd N = 4639 ± The ddition of n extr dose of TGF-P3 16 h before the irrdition, i.e. protocol involving five doses of TGF- 3 fforded similr levels of stem cell protection (Figure 3). The curve is highly significntly different from the sline/bsa curve (P <.1), nd the prmeters tht define the TGF-P3 curve re Do = ± 13.7 cgy, N= Figure 4 shows tht there is no sttisticlly significnt difference between the two modes of dministrtion of TGF-P3 (four doses v five doses). Tble 1 shows the levels of protection fforded by the TGF-P3 t ech individul dose, expressed s the rtio (protection fctor) of the levels of surviving crypts in the TGF-P3 group compred with the sline/bsa vehicle group. Also shown re exmples for which individul doses hve been repeted more thn once. The effect of incresing TGF-P3 dose per injection on rdition dmge induced by 14 Gy rdition is shown in Tble 2. An increse in crypt cell protection from 3.3 to 5.4 s the dose of TGF-P3 ws incresed from.5 to 1 gg per injection ws observed. Tble 3 shows the effect of vrying the injection regimens using stndrd dose per injection of 2.5,ug. The importnt observtion from this experiment is tht TGF-,3 dministrtion fter irrdition decreses the protective effect. A single injection 4 h fter irrdition with the stndrd dministrtion before irrdition reduces the protection fctor British Journl of Cncer (1997) 75(1), c.1.2 ) C 2 cn Ii 16 %N Figure 3 Intestinl crypt survivl curves for nimls pretreted with sline/bsa (five injections, protocol s Figure 1; () nd five injections of TGF-,3 (2.5 gg; *). A significnt difference between the two curves ws observed (P<.1) Cncer Reserch Cmpign 1997

4 TGF-,B3 protection in gut Tble 1 Intestinl crypt cell protection from y-ry irrdition by prior tretment with TGF-J3 Dose Protection fctor (Gy) Five injections of TGF-l3 Four injections of TGF-P3 c.1 -.Q * : CL o.1- The protection fctor represents the TGF-3 group men divided by the sline/bsa group men. A vlue of 4. mens tht four times more crypts survived in the TGF-,B3-treted group. The five injection groups were dministered 2.5 9g TGF-P3-24, -16, -8, -4, h before irrdition, while the four injection groups were dministered 2.5 9g TGF-j3-24, -8, -4, h before irrdition. Ech number represents the rtio from seprte group of six treted nd six sline nimls..1 - Figure 4 Intestinl crypt survivl curves compring the dt shown in Figures 2 nd 3 for nimls pretreted with either four (U) or five () injections of 2.5 9g of TGF-J3. The survivl curves do not differ significntly from ech other (P =.8846) from bout 4 to 1.6. Administrtion of TGF-P3 following rdition in fct significntly sensitized the crypts to rdition dmge. Here, only third of the crypts survived compred with the sline/bsa controls. To effectively reduce these complicting effects of orl nd hemopoietic dmge, we shielded the hed, thorx nd forelimbs of the niml in our survivl time experiments. Figure 5A shows the survivl time for mice receiving 14.5 Gy with nd without pretretment with TGF-f3. It cn be seen tht TGF-,3 lmost completely protects the mice from the effects of this dose of rdition. In the bsence of TGF-,3, pproximtely 65% of the nimls will die. Tble 2 The effect of TGF-f33 dose on crypt cell survivl Dose of TGF-3 (gg) Protection fctor The rtios of the crypt survivl vlues obtined in TGF-p3-treted nimls compred with those for sline/bsa-treted controls re presented. The dose of TGF-133 per injection ws vried. TGF-fi3 ws injected t -24, -8, -4 nd h before exposure to 14 Gy of gmm rys. The crypt cell protection rtio rose progressively with incresing dose. Figure 5B shows tht once the dose of rdition goes beyond criticl vlue (here 16 Gy), the levels of dmge re such tht TGF-,3 cn no longer protect the gstrointestinl mucos. Although more crypts survived (see Figure 3), their numbers re insufficient to mintin niml survivl. Tble 3 Protection of intestinl crypt cells s function of TGF-P3 dministrtion schedule Protocol Crypts per Men ± s.d. Width (gm) Size-corrected Protection circumference crypts per circumference fctor -24,-8,-4, 1.1, 17., 17.3, 6.4, 14.2 ± , ,-16,-8,-4, 17.9, 4.1, 1.9, ± , -24,-8, -4, 15.9, 14.8, 1.8, 14.2, 14.6 ± , , -8, -4,, , 5.6, 2.6, 7.1, 7.8, ± , +16, +24, ,.7, 1., 1.7, 1.1, ± Gy lone.7,.6, 3.5, 1.2, 2.3, ± Untreted nimls 93.6, 17.2, 13.2, 99.4, 97., ± Sline/BSA t -24,-16,-8, -4, 5.8, 2.8, 6.8, 3.6, 3.8, ± TGF-P3 (2.5,ug) ws dministered by i.p. injection. The i- symbol denotes time (h) before irrdition (14 Gy in ll cses) while the '+' symbol denotes time fter irrdition. ' time' denotes injections delivered immeditely fter irrdition. Protection fctor represents the TFG-P3 group men divided by the sline/bsa group men. The individul men vlues for the counts of surviving crypts for ech mouse re shown together with the width mesurements used for the size correction (Potten et l, 1981). Cncer Reserch Cmpign 1997 British Journl of Cncer (1997) 75(1),

5 1458 CS Potten et l - cn o 2 cn 1 5 A B 1', n CO) 5 e Sline/BSA Dys fter irrdition 1 2 Dys Figure 5 The survivl time of nimls exposed to 14.5 Gy (A) or 16 Gy (B) of 3 kvp X-rys delivered to the bdomen. (A) Approximtely 65% of the nimls pretreted with sline/bsa (-24, -8, -4, h) died between dys 3 nd 12, while only 5% of the nimls pretreted with TGF-3 succumbed to the effects of rdition. Two nimls in the TGF-j3 group died s consequence of the nesthesi delivered t the time of irrdition nd one niml died between dys 6 nd 7. The dt re presented in terms of percentge survivl. (B) Animl survivl time dt following dose of 16 Gy X-rdition (bdomen only). At this level of rdition dose nd dmge, dministrtion of TGF-f3 hs no effect on niml survivl DISCUSSION The trnsforming growth fctor bets re potent inhibitors of cell cycle progression in epithelil nd hemopoietic systems. TGF-,3 cts, t lest in prt, through inhibition of cdk-/cyclin-dependent kinse ctivity during the GI phse of the cell cycle (Polyk et l, 1994; Sherr nd Roberts 1995). We hve recently shown tht TGF-,B1 dministered over protrcted period of time cn significntly suppress the prolifertive ctivity in the smll intestinl crypts, the effect being prticulrly pronounced in the stem cell region (Potten et l, 1995). In ddition, TGF-ps cn hve the opposite effect on some cell types nd ct s indirect mitogens, prticulrly for mesenchyml cell types, nd exert effects on chemotxis nd on the synthesis nd degrdtion of extrcellulr mtrix proteins (Ignotz nd Mssgue 1986; Posthlethwite et l, 1987; Whl et l, 1987; Roberts nd Sporn, 1991). Here, we hve shown tht the propitious dministrtion of TGF-133 over 24-h period before exposure to cytotoxic gent (rdition) cn lter significntly the survivl of the crypt stem cells in vivo nd increse overll niml survivl. It is ssumed tht this is chieved s consequence of reversible TGF-p3- induced rrest of crypt stem cells cycling, thus rendering them more resistnt. TGF-fi1 or -2 hs been shown to reversibly inhibit prolifertion of bone mrrow stem cells nd lso to protect mice from the effects of 5-FU over the period of 2-2 dys (Grzegorzewski et l, 1994). Our stndrd reference delivery protocol involved four injections of 2.5 jg per injection per mouse delivered 24 h preceding irrdition (-24, -8, -4, h), with the finl injection being delivered immeditely fter the rdition exposure. Subtle vritions of this delivery regimen, by extending the dministrtion time or dministering evenly spced doses of TGF-f33, hd little effect on the levels of protection. Incresing the dose per injection hd smll beneficil effect. The dministrtion of some of the TGF-,3 injections fter exposure to rdition brogted the positive effect of TGF-,3 pretretment. Administrtion of the TGF-P3 following irrdition resulted in mrked sensitiztion. These dt re consistent with the view tht TGF-P33 ws inhibiting stem cell cycle progression, wheres continued dosing fter irrdition would be predicted to inhibit the regenertive process. The size of the crypts, s determined by their width mesurement, suggests tht TGF-133 ws ffecting the overll cellulrity. Pretretment with TGF-3 results in crypts 3-4 dys fter irrdition tht hve size slightly greter thn observed in nimls pretreted with sline/bsa (Tble 3). This would be expected if the TGF-3 enbled extr stem cells to survive per crypt. Continued cell cycle rrest by post-irrdition tretment with TGF-,3, s would be predicted, tended to result in slightly smller crypts, nd this demonstrtes the importnce of the crypt size correction process (Potten et l, 1981). There re indictions tht the stem cells in the crypts show slightly stronger circdin rhythm thn the mjority of the crypt cells nd hve their pek DNA synthetic ctivity centred round 3. h (Potten et l, 1977). It is lso believed tht the stem cells hve cell cycle durtion of pproximtely 24 h (Potten, 1995). Thus, delivering TGF-,3 over period of 24 h before dose of rdition delivered t 3. h might be expected to hve the mximum effect in preventing cells entering S, i.e. in reducing the number of DNA synthesizing cells in the stem cell comprtment t the time of irrdition. It remins to be seen whether other protocols with different spcings between the TGF-,3 injections might be more effective. The current experiments, in which the rdition ws delivered t 3. h, resulted in better levels of protection thn were observed in erlier preliminry experiments in which the time of irrdition tended to be between 9. nd 12. h. The well-being of the niml, nd similrly of ptient undergoing cncer therpy tretment, will depend on the competitive interction between cellulr depletion processes nd cellulr regenertion processes in the gstrointestinl trct. If the former re too severe, or the ltter too slow, the consequence is loss of mucosl integrity, which ultimtely results in the symptoms of the gstrointestinl rdition syndrome. If these symptoms re too severe, the niml will die from gstrointestinl dmge, nd this chrcteristiclly occurs over the period of bout dys The survivl of intestinl crypts is determined by the level of British Journl of Cncer (1997) 75(1), Cncer Reserch Cmpign 1997

6 TGF-,B3 protection in gut 1459 survivl of individul clonogenic stem cells within ech crypt. The survivl nd well-being of the niml depends ultimtely on the levels of surviving crypts (Hendry et l, 1983). Following whole-body irrdition, survivl of nimls is influenced in complicted fshion by dmge induced in the orl mucose nd in the hemopoietic system. The ltter is inherently more sensitive thn the gstrointestinl trct. Deths ttributed to bone mrrow dmge usully occur lter thn 12 dys. In order to reduce the consequences of this orl nd hemopoietic dmge, we chose to irrdite only the bdomens of the nimls in the survivl study. The survivl time experiment ws used s prdigm for ptient well-being in cncer tretment sitution, nd these experiments cn be viewed independently of the crypt survivl studies. However, the survivl curves shown in Figures 2 nd 3 show tht TGF-P3 cn hve firly drmtic effect on the frction of crypts surviving. The TGF-,B3 tretment effectively prevents dmge equivlent to 2 Gy of rdition, nd the consequences of this cn be seen very drmticlly in the survivl time study illustrted in Figure SA - in the bsence of TGF-j3 65% of the nimls die fter 14.5 Gy, while in the TGF-,B3-treted group only 5% die. If the dose is rised to 16 Gy, only bout.5% of the crypts survive in the bsence of TGF-P3 (Figure 1). TGF-P3, my in principle, protect some clonogenic cells nd rise this to bout 2.5%, but this is still t level of dmge tht results in niml deth (Figure 5B). The principles of these experiments my hve mjor clinicl implictions in terms of their potentil for improving the qulity of life of cncer therpy ptients. Furthermore, they my possibly offer the opportunity of dose escltion nd, hence, improvements in cure rte. However, there re mny questions still to be resolved. We hve chosen to use rdition s model cytotoxic for which the dose nd dose distribution cn be firly precisely controlled. We need to demonstrte tht TGF-i3 cn protect the intestinl epithelium ginst other forms of cytotoxic gents. Cncer chemotherpy involves the use of multiple gents delivered repetedly over protrcted period. There re no dequte niml models for such combintion therpies. We re currently developing such models nd the efficcy of TGF-,3 tretment within this context needs to be tested. Finlly, there is the question of to wht extent cells in tumours my be protected by TGF-j3 long with the norml tissue. Although this is not fully resolved, there is strong evidence indicting tht tumour cells re less responsive to the cell cycle progression inhibition of TGF-i3 by brogtion of the signlling pthwys, leding to the phosphoryltion of the retinoblstom (Rb) gene product (Sherr nd Roberts 1995). ACKNOWLEDGEMENTS This work hs been supported by the Cncer Reserch Cmpign (UK) nd by Oncogene Science (Uniondle, NY, USA). We re grteful to the stff of Lbortory 19 nd the histology lbortory for their continued vluble ssistnce. Mny members of the Deprtment of Epithelil Biology helped with these experiments for which we re extremely grteful. We thnk Arthur Bruskin for helpful suggestions nd comments during the course of these studies. REFERENCES Chbner BA (1993) Anticncer drugs In Cncer, Principles nd Prctice of Oncology, 4th edn, DeVit VT, Hellmn S nd Rosenberg SA. (eds), pp , Lipincott: Phildelphi Geng Y nd Weinberg RA (1993) Trnsforming growth fctor 3 effects on expression of G1 cyclins nd cyclin-dependent kinses. Proc Ntl Acd Sci 9: Grzegorzewski K, Ruscetti FW, Usui N, Dmi G, Longo DL, Crlino HA, Keller JR nd Wiltrout RH (1994) Recombinnt Trnsforming Growth Fctory l,, nd P2 protected mice from cutely lethl doses of 5-Fluorourcil nd doxorubicin. 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