Cholecystokinin (CCK) and CCK receptor expression by human gliomas:evidence for an autocrine/paracrine stimulatory loop

Cholecystokinin (CCK) is a gut-brain peptide has been described to be able to induce mitosis according to recent studies. Additionally, conflicting data has been published on whether tumours of the central and peripheral nervous system in general, and gliomas in particular, express CCK receptors. In the present in vitro study we employed reverse transcription followed by the polymerase chain reaction (RT-PCR) to investigate whether mRNA for CCK-A and CCK-B receptors as well as CCK peptide itself is present in primary human gliomas and the U-87 MG GBM cell line. The data show that 14/14 (100%) of the primary gliomas exhibited mRNA expression for the CCK peptide gene and the B receptor including the U-87 MG cells, whereas, only 2/14 (14%) showed presence of the CCK-A receptor. The presence of CCK receptors together with CCK peptide expression itself suggests presence of an autocrine loop controlling glioma cell growth. In support of this conclusion, a neutralizing antibody against the CCK peptide exhibited a dose dependent inhibition of cell growth whereas, antagonists to CCK caused a dose depend inhibition of exogenous stimulated glioma cell growth in vitro, via the CCK-B receptor which is PKC activated. Assessment of apoptosis and proteasome activity were undertaken and we report that treatment with CCK antagonists decreased proteasome and increased caspase-3 activity. These data indicate that CCK peptide and CCK-B are abundant in human gliomas and they act to stimulate cell growth in an autocrine manner, primarily via the high affinity CCK-B receptor, which was blocked by antagonists to CCK, perhaps via apoptosis.

Molecular biology and biochemistry of brain tumours

ßElucidating some molecular mechanisms and biochemistry of brain tumours is an important step towards the development of adjuvant medical therapies. The present study concentrates on cholecystokinin (CCK), a gut-brain peptide that has been described to be able to induce mitosis of rat gliomas as well as hormone secretion by the anterior pituitary, via the CCK-B receptor. The significance of a polymorphism in the growth hormone releasing hormone (GHRH) receptor (GHRH-R) gene was also determined. Finally, defects in the ß-catenin gene, an important component of the developmental pathway, in a sub-set of craniopharyngiomas were investigated. Reverse transcription-polymerase chain reaction (RT-PCR), restriction digestion analysis and direct sequencing demonstrated expression of CCK peptide itself and its A and B receptors by human gliomas, meningiomas and pituitary tumours. CCK peptides stimulated growth of cultured gliomas and meningiomas as well as in vitro hormone secretion [growth hormone (GH), luteinizing hormone (LH) and follicle stimulating hormone (FSH)] by human pituitary tumours. These biological effects were reduced or abolished by CCK antagonists. In addition, an antibody to CCK reduced mitosis by gliomas and meningiomas, and the same antibody inhibited hormone secretion by cultured human pituitary tumours. CCK peptides stimulated phosphatidylinositol (PI) hydrolysis, indicating coupling of the CCK receptors to phopsholipase C. Cyclic AMP was unaffected. In addition, caspase-3 activity was significantly and markedly increased, whilst proteasome activity was decreased. Taken together, these results may indicate an autocrine/paracrine role of CCK in the control of growth and/or functioning of gliomas, meningiomas and pituitary tumours. Primer induced restriction analysis (PIRA) of a rarer and alternative polymorphism in the GHRH-R receptor, in which Thr replaces Ala at codon 57, in human GH-secreting pituitary tumours was investigated. Whilst the rarer form correlated with an increased response of the pituitary cells to GHRH in vitro, allele distribution studies revealed that it is unlikely that the polymorphism contributes to increased risk of developing GH-secreting tumours and therefore acromegaly. Further findings of this study, using PCR and direct sequencing, were the demonstration of an association between b-catenin gene alterations and craniopharyngiomas of the adamantinomatous type. Since this gene product is involved with development, these results suggest that p-catenin mutations may contribute to the initiation and subsequent growth of congenital adamantinomatous craniopharyngiomas.

Studies on the mechanism of action on antitumour imidazotetrazinones

The imidazotetrazinones are clinically active antitumour agents, temozolomide currently proving successful in the treatment of melanomas and gliomas. The exact nature of the biological processes underlying response are as yet unclear.This thesis attempts to identify the cellular targets important to the cytotoxicity of imidazotetrazinones, to elucidate the pathways by which this damage leads to cell death, and to identify mechanisms by which tumour cells may circumvent this action. The levels of the DNA repair enzymes O6-alkylguanine-DNA-alkyltransferase (O6-AGAT) and 3-methyladenine-DNA-glycosylase (3MAG) have been examined in a range of murine and human cell lines with differential sensitivity to temozolomide. All the cell lines were proficient in 3MAG despite there being 40-fold difference in sensitivity to temozolomide. This suggests that while 3-methyladenine is a major product of temozolomide alkylation of DNA it is unlikely to be a cytotoxic lesion. In contrast, there was a 20-fold variation in O6-AGAT levels and the concentration of this repair enzyme correlated with variations in cytotoxicity. Furthermore, depletion of this enzyme in a resistant, O6-AGAT proficient cell line (Raji), by pre-treatment with the free base O6-methylguanine resulted in 54% sensitisation to the effects of temozolomide. These observations have been extended to 3 glioma cell lines; results that support the view that the cytotoxicity of temozolomide is related to alkylation at the O6-position of guanine and that resistance to this drug is determined by efficient repair of this lesion. It is clear, however, the other factors may influence tumour response since temozolomide showed little differential activity towards 3 established solid murine tumours in vivo, despite different tumour O6-AGAT levels. Unlike mitozolomide, temozolomide is incapable of cross-linking DNA and a mechanism by which O6-methylguanine may exert lethality is unclear. The cytotoxicity of the methyl group may be due to its disruption of DNA-protein interactions, or alternatively cell death may not be a direct result of the alkyl group itself, but manifested by DNA single-strand breaks. Enhanced alkaline elution rates were found for the DNA of Raji cells treated with temozolomide following alkyltransferase depletion, suggesting a relationship between O6-methylguanine and the induction single-strand breaks. Such breaks can activate poly(ADP-ribose) synthetase (ADPRT) an enzyme capable of rapid and lethal depletion of cellular NAD levels. However, at concentrations of temozolomlde relevant in vivo little change in adenine nucleotides was detected in cell lines, although this enzyme would appear important in modulating DNA repair since inhibition of ADPRT potentiated temozolomide cytotoxicity in Raji cells but not O6-AGAT deficient GM892A cells. Cell lines have been reported that are O6-AGAT deficient yet resistant to methylating agents. Thus, resistance to temozolomide may arise not only by removal of the methyl group from the O6-position of guanine, but also from another mechanism involving caffeine-sensitive post-replication repair or mismatch repair activity. A modification of the standard Maxam Gilbert sequencing technique was used to determine the sequence specificity of guanine-N7 alkylation. Temozolomide preferentially alkylated runs of guanines with the intensity of reaction increasing with the number of adjacent guanines in the DNA sequence. Comparable results were obtained with a polymerase-stop assay, although neither technique elucidates the sequence specificity of O6-guanine alkylation. The importance of such specificity to cytotoxicity is uncertain, although guanine-rich sequences are common to the promoter regions of oncogenes. Expression of a plasmid reporter gene under the control of the Ha-ras proto~oncogene promoter was inhibited by alkylation with temozolomide when transfected into cancer cell lines, However, this inhibition did not appear to be related to O6~guanine alkylation and therefore would seem unimportant to the chemotherapeutic activity of temozolomide.