Preoperative low dose NSAID treatment influences the genes for stemness, growth, invasion and metastasis in colorectal cancer.

Preclinical data, and an increasing list of clinical investigations, show anti-inflammatory agents to favourably influence the biology of colorectal tumor. We have earlier reported on re-expression of activated immune cells after three days preoperative treatment of patients with colorectal carcinoma, randomized to receive oral NSAID (indomethacin or celebrex). Antisecretory prophylaxis (esomeprasol) was provided to all patients and served as sham treatment. Concomittant to MHC locus activation, Prominin1/CD133, a marker associated with stemness and poor prognosis in several solid tumors, was downregulated. The aim of the present study was to evaluate expression of additional regulators belonging to the stem cell niche, OCT4, SOX2 and BMP7, as well as some microRNAs, reported to act as tumor suppressors or oncomiRs. Peroperative tumor biopsies were analyzed by microarrays, quantitative real-time PCR and immunohistochemistry (IHC). The stem cell master regulator SOX2 was increased by NSAIDs (p<0.01), as well as the tumor suppressor miR-630 (p<0.01), while BMP7, a marker for poor prognosis in CRC, was downregulated by NSAID (indomethacin, p<0.02). The upregulation of SOX2, but not of its heterodimer binding partner OCT4, could imply a negative feed-back loop, with a switch­off for stemness preservation of tumor cells. This is supported by the overall evaluation of gene expression profiles with subsequent events, indicating less aggressive tumors following NSAID treatment.

Downregulation of Prominin 1/CD133 expression in colorectal cancer by NSAIDs following short-term preoperative treatment.

Expression of Prominin 1/CD133 is associated with poor prognosis and chemoresistance in several types of solid tumors. The aim of the present study was, therefore, to evaluate Prominin 1/CD133 expression in colorectal carcinoma after short-term preoperative treatment by non-steroidal anti-inflammatory drugs (NSAIDs). Patients aimed at curative operation for colon cancer were randomized to receive NSAIDs (indomethacin 50 mg x2 or celecoxib 100 mg x2) three days preoperatively. Antisecretory prophylaxis (esomeprasol 40 mg x1) was provided to all patients and served as sham intake. CD133 expression in tumor tissue was also assessed in tumors from Dukes' B patients selected for either long or short postoperative survival. No patients received perioperative chemoradiotherapy. Tumor tissue was collected at surgery for quantification of mRNAs (Prom1 and Wnt4) by qPCR. Immunohistochemistry stained for CD133, Ep-CAM, CD34 and CD45. PGE2 content in tumor tissue was determined. Transcript of CD133 in tumor tissue was lower in patients treated with NSAIDs (0.28 ± 0.07 vs. 0.51 ± 0.08; p<0.03) as well as some other stem cell-related transcripts. In treated patients 36% of all tumors stained positive for CD133 compared to 71% in sham-treated control patients (p<0.05). Short survivors with Dukes' B tumors displayed 78% CD133 expression as compared to 33% of tumors in long-term survivors (p<0.002). Tumor tissue PGE(2) content was negatively related to patient survival. Our results show that short-term preoperative NSAID treatment downregulates colon cancer tissue expression of Prominin 1/CD133, a stem cell marker indicative of survival prognosis as confirmed.

Sexual function after failed ileal pouch-anal anastomosis.

BACKGROUND AND AIMS: Failure of ileal pouch-anal anastomosis (IPAA) occurs in around 10% of the patients. Compared to patients with functioning pouches, health related quality of life is deteriorated after failure. Sexual function in patients with pouch failure is however poorly studied. The aim was to study sexual function in patients with pelvic pouch failure; patients with functioning pouches were used as controls. The hypothesis was that patients with pouch failure have worse sexual function. METHODS: 36 patients with pouch failure were compared with 72 age and sex-matched controls with ulcerative colitis and functioning pouches. The patients answered a set of questionnaires concerning sexual function (Female Sexual Function Index [FSFI] and International Index of Erectile Function [IIEF]), body image (BIS-scale) and health-related quality of life (SF-36). RESULTS: Both women and men with pouch failure scored lower than controls in the FSFI and IIEF questionnaires. However, none of the observations were statistically significant. The scores in the failure group (for both sexes) were below the cut-off level for sexual dysfunction. Scores for the BIS instrument were significantly lower for both sexes in the failure group. Women and men in the failure group scored lower than the controls in all domains of the SF-36, however statistically significant only for the social function domain in men. CONCLUSIONS: The hypothesis, that a failed IPAA is associated with worse sexual function, was not confirmed. Compared to patients with functioning pouches, patients with pouch failure have inferior body image.

Is acetylcholine a signaling molecule for human colon cancer progression?

OBJECTIVE: Non-neuronal acetylcholine (ACh) has been suggested to be a mediator for the development of various types of cancer. We analyzed a possible role for this molecule in carcinogenesis and/or progression of human colon cancer, in patient biopsies harvested from the colon during surgery. We addressed whether ACh synthesis (by choline acetyltransferase) and/or degradation (by ACh esterase), as well as the expression of the α7-subtype of the nicotinic ACh receptors, and the peptide ligand at the α7 receptors, secreted mammalian Ly6/urokinase-type plasminogen activator receptor-related protein-1, respectively, are deranged in tumor tissue as compared with macroscopically tumor-free colon tissue. METHODS: A total of 38 patients were grouped for analysis based on their respective Dukes stage (either Dukes A + B or C + D). A mucosal tissue sample was harvested from macroscopically tumor-free colon tissue (i.e. control tissue), as well as from the tumor, and protein lysates were prepared for quantitative Western blotting. Full-thickness specimens were taken for immunohistochemistry. RESULTS: For all the above named markers, there was a significant difference between control and tumor tissue with regard to protein levels, and there was, in addition, a significant difference in protein levels between the Dukes A + B and C + D groups. CONCLUSION: The current findings may suggest a role for ACh in colon carcinogenesis/cancer progression; the data obtained could have prognostic and/or therapeutic significance for this disease.

A pharmacological analysis of the cholinergic regulation of urokinase-type plasminogen activator secretion in the human colon cancer cell line, HT-29.

Urokinase-type plasminogen activator (uPA) is an important factor for tumour cell invasion and metastasis. We recently showed that acetylcholine is an autocrine/paracrine growth factor for the human colon cancer cell line, HT-29, in part via the α7 subtype of the nicotinic acetylcholine receptors. In the current study, we investigated whether acetylcholine participates in the regulation of the protein expressions of also uPA and its receptor (uPAR) in the HT-29 cell line. Such were investigated by immunocytochemistry and Western blotting, and quantitation of uPA secretion was undertaken by ELISA. Stimulation of the cells for 24h with nicotine caused increased uPA secretion with peak effect (78% above the control) occurring at a nicotine concentration of 10nM. This effect was markedly inhibited by α-Bungarotoxin, thus showing the involvement of α7 nicotinic acetylcholine receptors. Basal uPA secretion was found to be partly dependent on ongoing activation of nicotinic receptors, suggesting tonic production of acetylcholine. Conversely, there was no cholinergic influence on the expression of uPAR. The current findings demonstrate novel aspects of receptor-mediated regulation of tumour metastatic potential via uPA secretion. This may suggest future pharmaceutical strategies in treatment of colorectal cancer.

Genes with relevance for early to late progression of colon carcinoma based on combined genomic and transcriptomic information from the same patients.

BACKGROUND: Genetic and epigenetic alterations in colorectal cancer are numerous. However, it is difficult to judge whether such changes are primary or secondary to the appearance and progression of tumors. Therefore, the aim of the present study was to identify altered DNA regions with significant covariation to transcription alterations along colon cancer progression. METHODS: Tumor and normal colon tissue were obtained at primary operations from 24 patients selected by chance. DNA, RNA and microRNAs were extracted from the same biopsy material in all individuals and analyzed by oligo-nucleotide array-based comparative genomic hybridization (CGH), mRNA- and microRNA oligo-arrays. Statistical analyses were performed to assess statistical interactions (correlations, co-variations) between DNA copy number changes and significant alterations in gene and microRNA expression using appropriate parametric and non-parametric statistics. RESULTS: Main DNA alterations were located on chromosome 7, 8, 13 and 20. Tumor DNA copy number gain increased with tumor progression, significantly related to increased gene expression. Copy number loss was not observed in Dukes A tumors. There was no significant relationship between expressed genes and tumor progression across Dukes A-D tumors; and no relationship between tumor stage and the number of microRNAs with significantly altered expression. Interaction analyses identified overall 41 genes, which discriminated early Dukes A plus B tumors from late Dukes C plus D tumor; 28 of these genes remained with correlations between genomic and transcriptomic alterations in Dukes C plus D tumors and 17 in Dukes D. One microRNA (microR-663) showed interactions with DNA alterations in all Dukes A-D tumors. CONCLUSIONS: Our modeling confirms that colon cancer progression is related to genomic instability and altered gene expression. However, early invasive tumor growth seemed rather related to transcriptomic alterations, where changes in microRNA may be an early phenomenon, and less to DNA copy number changes.

Patient-centred attitudes among medical students: gender and work experience in health care make a difference.

BACKGROUND: Previous studies of medical students' patient-centred attitudes show a decline across undergraduate education and overall higher scores for female students. AIM: To assess undergraduate students' patient-centred attitudes at various stages of education and to explore possible associations between attitudes and age, gender and work experience in health care. METHODS: In autumn 2005, medical students in Gothenburg (n = 797) were asked to answer Patient-Practitioner Orientation Scale (PPOS), a validated instrument exploring attitudes towards the doctor-patient relationship. Data including gender, age, current term and students' work experience in health care were collected. RESULTS: Of 797 students 600 (75%) answered the questionnaire. No decrease of students' PPOS score across the curriculum was observed. PPOS scores from female students were higher compared to males (p < 0.0001) and female scores were significantly higher in the later terms compared with earlier (p = 0.0011). Female students had more experience from working in health care (p = 0.0023). Extended work experience was associated with higher PPOS only among females (p = 0.0031). CONCLUSION: No decline of students' patient-centred attitudes may indicate an ongoing shift. Gender differences in patient-centred attitudes were reproduced. Work experience in health care presents a new gender difference. These gender differences should be considered when training patient-centred attitudes and skills.

Receptor and enzyme expression for prostanoid metabolism in colorectal cancer related to tumor tissue PGE2.

Prostaglandins support progression of colorectal cancer by several mechanisms. This conclusion is based on epidemiological and drug intervention long-term studies or retrieved from animal and cell culture experiments. The aim of the present study was to map receptor and enzyme expression for prostanoid metabolism in the presence of high or low PGE2 content within colon cancer tissue at primary tumor operation and after short-term preoperative provision of non-steroidal anti-inflammatory drug (NSAID). Twenty-three unselected patients with colon cancer were randomly selected to receive indomethacin (NSAID) or sham treatment for 3 days before surgery. Normal colon and tumor tissue were collected at operation for RNA extraction. Tissue PGE2 levels were measured by radioimmunoassay. Gene expression was quantified by microarray and real-time PCR. COX-1 expression increased proportionally to COX-2 expression in colon cancer tissue from untreated patients. Indomethacin reduced PGE2 content in normal and tumor tissue with subsequently decreased IP, HPGD and PPARgamma receptor expression in both tumor and normal colon tissue, while subtype EP1-4 receptors were not significantly influenced by indomethacin treatment. MPGES-1 expression was not related to overall PGE2 content in tumor and colon tissue, but decreased significantly in normal tissue during indomethacin exposure. Reduction of tumor tissue PGE2 was related to significant alteration in expression of several hundred genes indicating decreased cell cycling and increased apoptosis during indomethacin treatment, probably related to upregulation of acute phase reactants in tumor tissue. Increased prostanoid activity in colon cancer tissue is related to cross-talk between tumor and stroma cells.

Differential prognostic impact of uPA and PAI-1 in colon and rectal cancer.

BACKGROUND AND OBJECTIVES: Degradation of extracellular matrix is important for tumour growth and invasion, which in part is regulated by the plasminogen activation system. The aim of the study was to evaluate the protein expression of urokinase plasminogen activator (uPA) and plasminogen-activating inhibitor-1 (PAI-1) in plasma, tumour-free mucosa and tumour tissue regarding their prognostic value in colon and rectal cancer. METHODS: Patients (n = 221) undergoing surgery for colorectal cancer were prospectively included. Samples were assayed by ELISA technique. RESULTS: PAI-1 in tumour tissue (p = 0.006), plasma (<0.0001) and uPA in tumour-free mucosa (p = 0.006) were associated with survival in rectal cancer in univariate analysis. An uPA expression level below 1.1 ng/mg (log rank test, p < 0.0001) in tumour-free mucosa was associated with poor survival in rectal cancer. This was true also for patients without disseminated disease (M(0), p = 0.02). PAI-1 in plasma correlated with metastatic disease (p < 0.0001). uPA and PAI-1 were not associated with survival in either tumour tissue, mucosa or plasma in patients with colon cancer. CONCLUSIONS: uPA and PAI-1 have a differential prognostic impact in colon and rectal cancer. Preoperative mucosal uPA and plasma PAI-1 protein expression could possibly be used as prognostic factors in rectal cancer.

Nicotine induced modulation of SLURP-1 expression in human colon cancer cells.

The secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein-1 (SLURP-1) is an endogenous ligand at the alpha 7 subunit of the nicotinic acetylcholine receptor (nAChR). SLURP-1 has anti-tumourigenic properties. In the current study, we demonstrate that the challenge of HT-29 human colon cancer cells with nicotine for 24 h to increase cell growth via the alpha 7nAChRs, caused a marked reduction of the protein expression of SLURP-1. We suggest that there is an interplay between acetylcholine and SLURP-1 in the HT-29 cells, both molecules serving as autocrine growth controlling ligands at the alpha 7nAChR, where acetylcholine regulates the release of SLURP-1.

Is acetylcholine an autocrine/paracrine growth factor via the nicotinic alpha7-receptor subtype in the human colon cancer cell line HT-29?

We used immunochemistry to demonstrate expression of acetylcholine's nicotinic alpha7-receptor subtype in human colon cancer cell line HT-29. Moreover, RT-PCR and immunochemistry showed that choline acetyltransferase and acetylcholine esterase, the enzymes responsible for acetylcholine synthesis and degradation, respectively, localise in HT-29 cells. Bromoacetylcholine bromide, an inhibitor of choline acetyltransferase, significantly attenuated basal cell growth. Our findings suggest that acetylcholine might serve as an autocrine/paracrine-or speculatively, even intracrine-signalling molecule in cell line HT-29, thus contributing to carcinogenesis/cancer progression.

Preoperative treatment with a non-steroidal anti-inflammatory drug (NSAID) increases tumor tissue infiltration of seemingly activated immune cells in colorectal cancer.

This study evaluates HLA gene expression and tumor infiltration by B-cells, macrophages, dendritic cells, T-helper and cytotoxic T-lymphocytes in response to short-term preoperative treatment with cyclooxygenase inhibitors. Patients with colorectal carcinoma were randomized to receive oral NSAID (indomethacin or celebrex) for three days preoperatively; controls received esomeprazol. Peroperative tumor biopsies and normal colon tissue were analyzed by microarray, quantitative PCR and immunohistochemistry. Efficacy of short-term systemic NSAID treatment was confirmed by measurement of PGE2 production in blood monocytes from healthy volunteers. NSAID treatment upregulated genes at the MHC locus on chromosome 6p21 in tumor tissue, but not in normal colon tissue, from the same patient. 23 of the 100 most upregulated genes belonged to MHC class II. HLA-DM, -DO (peptide loading), HLA-DP, -DQ, -DR (antigen presentation), granzyme B, H, perforin and FCGR3A (CD16) (cytotoxicity) displayed increased expression, as did CD8, a marker of cytotoxic T-lymphocytes, while HLA-A and -C expression were not increased by NSAID treatment. MHC II protein (HLA-DP, -DQ, -DR) levels and infiltration by CD4+ T-helper cells of tumor stroma increased upon NSAID treatment, while CD8+ cytotoxic T-lymphocytes increased in both tumor stroma and epithelium. Molecules associated with immunosuppressive T regulatory cells (FOXP3, IL-10) were significantly decreased in indomethacin-exposed tumors. Standard oral administration of NSAID three days preoperatively was enough to increase tumor infiltration by seemingly activated immune cells. These findings agree with previous information that high prostanoid activities in colorectal cancer increase the risk for reduced disease-specific survival following tumor resection.

Growth associated proteins in tumor cells and stroma related to disease progression of colon cancer accounting for tumor tissue PGE2 content.

Connections among specific proteins (Bax, Bcl-2, bFGF, COX-1, COX-2, E-cad, p15, p53, PCNA, TGFbeta3, TUNEL, vWF) in control of cell proliferation, apoptosis, cell adhesion, tumor vascularity and PGE2 content were evaluated in colon cancer as related to disease progression and survival. Tumor tissue and adjacent normal colon mucosa were obtained at curative resection in 22 patients. PGE2 concentrations were assessed in tumor tissue and tumor derived blood, splanchnic blood, peripheral venous blood and urine. Host inflammation was determined (CRP, ESR) in relationship to tumor differentiation and stage. Patients survived as expected according to Dukes A-D staging. Growth-related proteins correlated between tumor cells and stroma as well as between protein factors within tumor cells and tumor stroma. COX-2 predicted tumor tissue content of PGE2 (p<0.002), without reflection in tumor derived blood. Systemic inflammation was predicted by p15, TGFbeta3 and Bcl-2 in tumor tissue (p<0.001). p15 and vWF predicted reduced survival in ungrouped patients (p<0.02), while p15, PCNA, TGFbeta3 and vWF predicted reduced survival (p<0.0001) when patient grouping accounted for high tumor content of PGE2. Our results connect systemic inflammation and survival to COX-2 staining and increased PGE2 in colon cancer. Thus, it seems important to understand proximal signals behind upregulation of COX-2 and subsequent PGE2 production in certain tumor cells in colon cancer.

Functional expression of mu-opioid receptors in the human colon cancer cell line, HT-29, and their localization in human colon.

We have investigated the functional expression of mu-opioid receptors (MORs) in the human colon cancer cell line, HT-29. As revealed by immunocytochemistry, immunoreactivity was present in both the cytoplasm and nuclei of the cells. Challenge with morphine for 24 h (1 nM to 1 microM) barely affected cell proliferation, while the secretion of urokinase type plasminogen activator (a protease involved in invasion/metastasis) was markedly augmented by a concentration of 0.1 microM. Human colon cancer tissue from 14 consecutively operated patients was investigated by immunohistochemistry. MORs were found in the nuclei of colonocytes and immune cells of the lamina propria in tumor-free tissue. In tumor tissue, immunoreactivity was found in the membrane and often in the nuclei of tumor cells. The current findings suggest that morphine administration could affect tumor progression by interfering with, for example, invasive properties. Our demonstration of a nuclear expression of the MORs appears to be a novel finding.

Prostanoid receptor expression in colorectal cancer related to tumor stage, differentiation and progression.

INTRODUCTION: Alterations in eicosanoid metabolism is well established in a variety of malignant tumors, particularly colorectal carcinoma. Recent studies in our laboratory have emphasized a role for EP subtype receptors in progression of colorectal cancer and disease specific mortality. Therefore, the aim of the present study was to extend our knowledge to include additional receptor expression (DP1, DP2, FP, IP, TP) for prostanoids (PGD2, TXA2, PGF2alpha, PGI2) in relationship to tumor stage, differentiation and progression of colorectal cancer. MATERIAL AND METHODS: Total RNA from 62 tumors and adjacent normal colon tissue (n = 48) was extracted. Quantification of receptor expression was performed by realtime PCR and related to the expression of an appropriate housekeeping gene (GAPDH). Tumors were assessed according to Dukes A-D (stage I-IV). RESULTS: DP1, DP2, FP and IP receptor subtypes displayed significantly reduced overall expression in tumor tissue compared to normal colon tissue, while the TP receptor subtype showed significantly higher expression in tumor tissue. Overall expression of the prostanoid receptors in tumor tissue was not related to clinical indexes as tumor stage and tumor cell differentiation evaluated by multivariate analyses. Cultured colorectal cancer cell lines with low (HT-29) and high (HCA-7) intrinsic PGE2 production at confluent state did not express DP1 and IP receptor subtypes, but displayed low expression of DP2, FP and TP receptor subtypes. CONCLUSION: The results in the present study indicate imbalanced expression of prostanoid receptors in colorectal cancer compared to normal colon tissue without clear cut relationship to disease progression. Therefore, future studies should be performed on defined cells within the tumor tissue compartment determining whether any prostanoid receptor(s) is useful as a molecular target in treatment or prevention of colorectal cancer.

EP1-4 subtype, COX and PPAR gamma receptor expression in colorectal cancer in prediction of disease-specific mortality.

The importance of prostaglandins in tumor growth and progression is well recognized, including antineoplastic activities by cyclooxygenase (COX) inhibitors. Variation in treatment response to COX inhibition has questioned differences in expression of cell surface and nuclear membrane receptors among tumors with different disease progression. The purpose of this study was to evaluate whether EP(1-4) subtype, PPAR gamma receptor and COX-1/COX-2 expression in colorectal cancer are related to tumor-specific mortality. Reverse transcription-polymerase chain reaction and immunohistochemistry were used to demonstrate expression and protein appearance in tumor tissue compared with normal colon tissue. EP(1) and EP(2) subtype receptor protein was highly present in tumor cells, EP(3) occurred occasionally and EP(4) was not visible. PPAR gamma, EP(2) and EP(4) mRNA were significantly higher in normal colon tissue compared with tumor tissue, without any distinct relationship to Dukes A-D tumor stage. Multivariate analyses indicated that increased tumor tissue EP(2) and COX-2 expression predicted poor survival (p<0.001). COX-1 expression was significantly higher than COX-2 expression in normal colon tissue. Average COX-2 mRNA was not increased in tumor tissue compared with normal colon. However, most tumor cells stained positive for COX-2 protein, which was low or undetectable in normal mucosa cells. COX-1 protein was preferentially visible in stroma. EP(1-4) subtype receptor mRNAs were generally positively correlated to both COX-1 and COX-2 in tumor tissue, but not in normal colon. Our results imply that both prostaglandin production (COX-2) and signaling via EP(1-4) subtype receptors, particularly EP(2), predict disease-specific mortality in colorectal cancer.

Tumor genome wide DNA alterations assessed by array CGH in patients with poor and excellent survival following operation for colorectal cancer.

Genome wide DNA alterations were evaluated by array CGH in addition to RNA expression profiling in colorectal cancer from patients with excellent and poor survival following primary operations. DNA was used for CGH in BAC and cDNA arrays. Global RNA expression was determined by 44K arrays. DNA and RNA from tumor and normal colon were used from cancer patients grouped according to death, survival or Dukes A, B, C and D tumor stage. Confirmed DNA alterations in all Dukes A - D were judged relevant for carcinogenesis, while changes in Dukes C and D only were regarded relevant for tumor progression. Copy number gain was more common than loss in tumor tissue (p < 0.01). Major tumor DNA alterations occurred in chromosome 8, 13, 18 and 20, where short survival included gain in 8q and loss in 8p. Copy number gains related to tumor progression were most common on chromosome 7, 8, 19, 20, while corresponding major losses appeared in chromosome 8. Losses at chromosome 18 occurred in all Dukes stages. Normal colon tissue from cancer patients displayed gains in chromosome 19 and 20. Mathematical Vector analysis implied a number of BAC-clones in tumor DNA with genes of potential importance for death or survival. The genomic variation in colorectal cancer cells is tremendous and emphasizes that BAC array CGH is presently more powerful than available statistical models to discriminate DNA sequence information related to outcome. Present results suggest that a majority of DNA alterations observed in colorectal cancer are secondary to tumor progression. Therefore, it would require an immense work to distinguish primary from secondary DNA alterations behind colorectal cancer.

Expression of P2Y2 purinoceptors in MCG 101 murine sarcoma cells, and HT-29 human colon carcinoma cells.

We investigated how agonists at purinoceptors may affect tumour cell metabolism. This was investigated in vitro in tumour cell lines by microphysiometry, which method monitors extracellular acidification rate (ECAR), on-line. The cell lines investigated were the murine sarcoma, MCG 101, and the human colon cancer, HT-29. In MCG 101, adenosine-5'-triphosphate (ATP) or uridine-5'-triphosphate (UTP) caused a concentration-dependent increase in ECAR, most likely due to the ligation of P2Y(2) receptors, which response was blocked by suramin. In HT-29, ATP or UTP elicited a concentration-dependent, biphasic change in ECAR (increase/decrease). The pharmacological analysis suggests the involvement of P2Y(2) receptors, although other P2 receptor subtypes cannot be entirely excluded. This biphasic response to UTP or ATP was resistant to suramin. The expression of P2Y(2) receptors was demonstrated in both cell lines by immunocytochemistry and Western blot. The current study, thus, shows the functional and morphological expression of a purinoceptor subtype with partly different effects on metabolism in two different tumour cell lines.