Abstracts 71

DES (ppb) XPA XPA/p53 -

WT

0 1500 0

Primary tumours M F M F M Testicular interstitial 0 1 0

cell adenoma Osteosarcoma 0 0 1 0 0 Lymphoma 0 0 1 0 0 Pituitary adenoma 0 0 5 3 0 Mammary carcinoma 0 0 Phaeochromocytoma 0 0 0 0 0 Brain glioma 0 0 0 0 0 Cervical fibrosarcoma 0 0 Total of tumour- 0 0 8 3 0

bearing animals

1500 0 1500

F M F M F M F 4 0 1

0 4 2 0 0 0 0 0 0 2 0 0 1 0 0 220022 1 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 1 12 7 0 0 4 2

Transcriptional Response to Glutathione Depletion

H. Powell, N.R. Kitteringham, Y. Clement, C. O’Donnell, C. Goldring, M. Pirmohamed. B.K. Park Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool L69 3GE, UK

Chemical and oxidative stress, characterised by depletion of reduced glutathione (GSH), results in a cellular response aimed at restoring GSH levels. Part of this response involves enhanced expression of the dimeric enzyme y-glutamylcysteine synthetase (y-GCS), the rate-limiting enzyme of GSH synthesis. Transcription of y-GCS subunit genes is in part regulated by the transcription factor AP-1 (Mulcahy and Gipp, 1995), a protein formed from dimers of the Fos and Jun families of proteins (Angel and Karin, 1991). with Jun homodimers being more active than Fos/Jun heterodimers (Sekhar et al., 1997). The stimulus for up-regulation of antioxidant genes in response to GSH depletion remains unresolved, with GSH levels themselves or the accumulation of GSH-conjugates being postulated (Shelton et al., 1986). We have therefore compared y-GCS mRNA and protein levels and factors regulating its expression following administration of either diethyl maleate (DEM), which depletes GSH by conjugate formation, or buthionine sulphoximine (BSO), which depletes GSH through inhibition of y-GCS, without conjugate formation. In addition, we have studied the expression of the alpha chain of the nascent polypeptide-as- sociated complex (c(-NAC), a recently discovered transcriptional co-activator of AP-1 in differentiating bone that preferentially enhances the activity of Jun homodimers (St-Arnaud and Quelo, 1998).

Male CD-l mice (30-40 g; n = 6 per group) were administered a single i.p. dose of either DEM (4.7 mmol/kg) or BSO (7.2 mmol/kg in saline). At 0.5, 1, 2, 3, 5 and 24 h, hepatic GSH was determined. Hepatic y-GCS, c-fos, c-jun and alpha-NAC mRNAs were measured by RT-PCR; AP-1 DNA-binding activity was determined by the electrophoretic mobility shift assay: and y-GCS and alpha-NAC protein levels were determined by Western blotting.

Hepatic GSH depletion of 60-70% was observed within 2 h following the administration of both DEM and BSO, with levels returning to pre-treatment values by 24 h. DEM treatment led to an increase in both y-GCS mRNA (3-fold) and protein levels (1.8-fold), which reflected concomitant rises in c-fos ancl c-jun mRNA levels, and AP-1 DNA binding activity. In contrast, no significant changes in either the transcription or expression of y-GCS were observed following the administration of BSO, despite

12 Abstracts

replenishment of GSH. Concurrent with this observation was the lack of enhanced AP-1 DNA binding activity and even a small decrease in c-jun mRNA levels with BSO. Interestingly, both treatments resulted in a fall in both mRNA and protein levels of a-NAC, suggesting that GSH itself regulates levels of this co-activator, although the physiological role of a-NAC in the liver is not known.

These studies suggest that GSH depletion alone may not be a sufficient stimulus for activation of y-GCS and that other stimuli, for example GSH conjugation, may be required. As such, further studies are required to unravel the increasingly complex regulation of GSH synthesis by transcription factors and various redox signalling pathways.

This work was supported by The Wellcome Trust. YC is in receipt of a Royal Society Travelling Fellowship. BKP is a Wellcome Principal Fellow.

Angel, P., Karin, M., 1991. Biochem. Biophys. Acta 1072, 129-157. Mulcahy, R.T., Gipp, J.J., 1995. Biochem. Biophys. Res. Commun. 209, 227-233. Sekhar, K.R., Meredith, M.J., Kerr, M.D., Soltaninassab, S.R., Spitz, D.R., Xu, Z.Q., Freeman, M.L., 1997. Biochem. Biophys. Res. Commun. 234, 5888593. Shelton, K.R., Egle, P.M., Todd, J.M., 1986. Biochem. Biophys. Res. Commun. 134, 492-498. St-Arnaud, R., Quelo, I., 1998. Front. Biosci. 3, 838-848.

Cytotoxic and DNA Binding Properties of New Bis-Naphthalimido Polyamine Derivatives

V. Rodilla, V. Pavlov, D. Hion and P. Kong Thoo Lin School of Applied Sciences, The Robert Gordon University, Aberdeen AB25 lHG, UK

The natural polyamines spermine, spermidine and putrescine have attracted considerable interest recent years due to their role in cell growth and differentiation (Tabor and Tabor, 1984). The inhibition of polyamine biosynthesis is generally associated with a decrease in cell proliferation and has been the primary focus of many antiproliferative studies. A more recent strategy has been to design polyamine analogues and derivatives capable of producing antiproliferative and cytotoxic effects (Pegg, 1988).

In this study the growth inhibitory effects of newly synthesised bis-naphthalimido polyamine derivatives against breast cancer MCF7 cells were investigated and their binding affinity to DNA was measured. The cytotoxicity of bis-naphthalimido spermidine (PK4) and spermine (PK3) were measured by using a standard MTT assay. The binding affinity of the new polyamine derivatives was investigated spectrophotometrically by determining the change in the midpoint (Tm) of the thermal denaturation curves of calf thymus DNA.

The results from the growth inhibitory assay (Table 1) show that both PK4 and PK3 are highly cytotoxic against MCF7 cells. These new bis-naphthalimido polyamines strongly stabilise the thermal helix + coil

Table 1

Growth inhibitory effects and DNA binding of the bis-naphthalimido polyamines

Compound TC,, (PM) ATm (“C)b

1:lO 1:s

PK4 1.38 29 7, 29

PK3 2.91 28 10, 28

a Drug concentration required to inhibit cell growth by 50% (mean value of at least three experiments).

b ATm = Tm(DNA+drugkTm(DNA) (mean value from three determinations for the [drug]:[DNA] ratio shown). Where two

values are shown this indicates a biphasic DNA melting profile.