Prostate tumours from an Asian population: examination of bax, bcl-2, p53 and ras and identification of bax as a prognostic marker.

Molecular studies have suggested that ethnicity may play a significant role in prostate tumorigenesis, but no information exists for groups other than Caucasian or Japanese patients. We examined 62 archival samples of prostate tumours from Asians of non-Japanese origin for the over-expression of p53, for the possible presence of mutated ras genes, for the overexpression of the bcl-2 and bax proteins, as well as directly for the presence of apoptotic cells by the TUNEL methodology. Gene lesions of both ras (0%) and p53 (3%) were rare. While bcl-2 expression was not observed in any sample, bax expression was noted in 76% of samples and was associated with a significantly worse patient prognosis both overall (P< 0.005) and specifically in Chinese patients (P< 0.02). Apoptotic cells were found in 61% of samples, and were significantly associated with the presence of bax expression (P = 0.002), but not patient survival. These results suggest that prostate tumours from non-Japanese Asians are genetically distinct from prostate tumour found in both Japanese and Caucasian patients, and that treatment modalities may need to be tailored for specific population groups.

HIT family genes: FHIT but not PKCI-1/HINT produces altered transcripts in colorectal cancer.

Forty-five colorectal adenocarcinomas were examined for alterations in the HIT family genes FHIT and PKCI-1/HINT by a combination of reverse transcriptase polymerase chain reaction and DNA sequencing. In all cases a single transcript corresponding to the reported sequence was detected using primers specific for the PKCI-1/HINT gene. In contrast multiple transcripts were detected using primers specific for the FHIT gene transcript. 6% (3/45) of tumours evinced no detectable expression of any FHIT transcript and a further 12% (6/45) produced only the normal full length transcripts. Ninety-six aberrant transcripts were characterized from the remaining tumours. Deviations from the normal full length sequence characterized included deletions, insertions of novel sequences, a point mutation as well as the usage of a putative alternate splice site in exon 10. Message variants were detected with approximately equal frequency in all tumour stages with the exception that templates with insertions were found solely in Dukes' stage B tumours (P < 0.001). With the exception of the putative alternate splice site, aberrant transcripts were not detected in matched normal mucosa. These results suggest that members of the HIT family of genes are only selectively involved in tumorigenesis and that perturbation of FHIT gene expression is an early event in colorectal tumorigenesis.

c-Ki-ras mutations in colorectal adenocarcinomas from a country with a rapidly changing colorectal cancer incidence.

We have examined the incidence of mutation of the c-Ki-ras proto-oncogene in colorectal adenocarcinomas from two different time periods, namely 1962-1966 and 1994-1996. The first cohort of samples consisted of formalin-fixed, archival paraffin block and represent the oldest colorectal cancer samples for which ras mutation has been examined, while the second cohort of tumours were fresh, flash-frozen samples representative of genetic events occurring in contemporary times. Analysis of mutation status was undertaken by a mismatch-specific oligonucleotide hybridization analysis of exon 1 of the c-Ki-ras proto-oncogene after amplification by the polymerase chain reaction. Mutations in codon 12 or 13 of c-Ki-ras were detected in 28% (14/50) of contemporary samples, a figure consistent with locally established mutation rates. In contrast no mutation was detected in any of the 18 samples from the earlier period, a result that is statistically significant (P = 0.007). Age-standardized rates of colorectal cancer in Singapore have seen a marked increase over the last 30 years, and for the first time we have shown that such an increase in colorectal cancer is associated, at least in part with an increase in incidence of a specific mutagenic change.

p53 point mutation and survival in colorectal cancer patients: effect of disease dissemination and tumour location.

The relationship between p53 point mutation and patient survival was examined in 328 colorectal cancer patients. Point mutation was detected in 51% (166/328) of cases and was associated with a poorer prognosis in univariate and multivariate analysis. However, subcohort analysis showed that this relationship was restricted to patients with lymphatic dissemination, patients without evident distant metastatic lesions at the time of presentation and in tumours confined to the distal colorectum. These results suggest that the utility of p53 point mutation as a specific, patient based prognostic marker may be restricted to certain classes of patients.

C-KI-RAS activation and the biological behaviour of proximal and distal colonic adenocarcinomas.

One hundred and forty colonic adenocarcinomas originating on the left side of the colorectum and 70 colorectal carcinomas originating on right side of the colorectum were examined for activating mutations of codons 12 and 13 of the C-KI-RAS proto-oncogene. Rates of mutation were significantly different (right colon 43%, 30/70 versus left colon 23%, 32/140; P = 0.0025). Adenocarcinomas from the left side of the colorectum showed a significant association between C-KI-RAS activation and tumour progression, including the presence of distant organ metastasis at the time of surgery (P = 0.0039), and during patient follow-up (P = 0.00027), whereas those from the right of the colorectum did not (P = 0.4 and P = 0.5, respectively). Mutation of the C-KI-RAS proto-oncogene was found to be associated with a significantly poorer patient prognosis on the left of the colorectum (P = 0.0001 by log rank analysis of Kaplan-Meier plots) but not on the right (P = 0.7). These results demonstrate that, not only is the timing and frequency of C-KI-RAS activation different between carcinomas originating on the left or right of the colorectum, but also that the biological consequences of such mutations may differ.

Molecular changes of colorectal cancer in Singapore.

Improved success in the management of colorectal cancer requires a better understanding of its development and biological behaviour. The key for this is molecular genetics. Gene changes parallel the multi-step changes in the adenoma-carcinoma sequence. Cancer results from a variable combination of defects in oncogenes, tumour suppressor, mutator and apoptotic genes. These changes are similar whether they occur in inherited disorders like adenomatous polyposis coli (APC) and hereditary non-polyposis colorectal cancer (HNPCC) or acquired cancer in the elderly. In Singapore, the c-myc and c-Ki-ras proto-oncogenes are found to be activated in 70% and 29% of tumours respectively. Allelic loss of chromosome 5q and 17p occurs in 25% and 70% of tumours respectively, while point mutation of the p53 tumour suppressor gene occurs in 50% of colorectal cancers. Both the frequency and the nature of the lesion occurring are compatible to the changes detected in Caucasian patients, suggesting common aetiological factors. The biological behaviour of colorectal adenocarcinomas is determined by the nature of defects or mutations in key genes such as the p53 tumour suppressor gene. Lymphatic spread is associated with the presence of point mutations and haematogenous spread is associated with loss of heterozygosity of p53. Survival is worse when conserved regions of the gene are mutated compared with those outside, and worst when codon 175 is mutated. Sensitivity to radiotherapy and chemotherapy is also determined by p53 mutation which controls apoptosis. Prognosis could now be individualised and with the prospect of gene therapy, molecular genetics will have a major impact on the management of colorectal cancer.

A comparison of the detection of activated c-Ki-ras genes by mismatch specific oligonucleotide hybridisation analysis and enriched PCR in colorectal adenocarcinomas.

We have examined 56 colorectal adenocarcinomas for activation of the c-Ki-ras proto-oncogene by two independent techniques, namely mismatch specific oligonucleotide hybridisation and an enriched PCR methodology. Biological significance of c-Ki-ras activation was assessed via Kaplan Meier analysis of patient survival. A total of 27% (15/56) of tumours were found to contain activated c-Ki-ras genes by mismatch specific oligonucleotide hybridisation and mutation was significantly associated with a poorer patient prognosis (p=0.018, log rank analysis). A total of 55% (31/56) of tumours were found to contain activated c-Ki-ras genes by enriched PCR, and again an association was noted between mutation and patient survival, but of a lower significance (p=0.029, log rank analysis). These results indicate that a high proportion of colorectal adenocarcinomas may contain low levels of activated c-Ki-ras genes, but this may be of limited biological significance.

Point mutation of the c-Ki-ras proto-oncogene and the p53 tumour suppressor gene in distal colonic adenocarcinomas.

One hundred and twenty-seven monochronous primary colorectal adenocarcinomas were examined for both activation of the c-Ki-ras proto-oncogene by point mutation in codons 12 or 13 and inactivation of the p53 tumour suppressor gene by point mutation in exons four through nine. All of the carcinomas originated in the distal half of the colorectum. Activation of the c-Ki-ras proto-oncogene was detected in 23% (29/127) of adenocarcinomas, and p53 inactivation was detected in 63% (80/127) of adenocarcinomas. Only 16% (20/127) of cases exhibited both genetic changes, and no association was noted between the occurrence of these two changes. Individually, both ras activation and p53 inactivation were associated with a poorer patient prognosis (p=0.0009 and p=0.0159 respectively). In a Cox regression analysis both ras activation and p53 inactivation contributed independently towards patient mortality. These results suggest that mutations exert their effect independently of each other, and that it is the cumulative mutational load that determines a patient's prognosis.

Association of activated proto-oncogenes ras and myc in colorectal carcinomas.

We have examined 60 colorectal carcinomas for activation of two proto-oncogenes, c-myc and c-Ki-ras. Over-expression of c-myc mRNA as determined by Northern analysis was found in 58% of cases (35/60). Activation of the c-Ki-ras gene by point mutations in codons 12 or 13 as determined by mismatch specific oligonucleotide hybridisation was found in 35% (21/60) of cases. There was a statistically significant association between activation of c-Ki-ras and over-expression of c-myc (P = 0.03), with 76% of tumours with an activated c-Ki-ras proto-oncogene showing over-expression of c-myc. The association was significant in left-sided colorectal tumours (P = 0.03) but not right-sided (P = 0.5). However, whereas only 59% of left side tumours showed at least one of the two changes (ras activation only, or myc activation only or both), 93% of right side tumours showed at least one of the changes (P = 0.01). Twenty-two percent of left side tumours showed both changes compared with 35% of right side tumours, although this result did not achieve significance (P = 0.2). These results suggest that in left-sided colorectal tumours ras and myc cooperate, as established in vitro, to produce neoplastic transformation while different pathway(s) are involved in right-sided tumours.

Autoradiographic localization and quantitation of beta 1- and beta 2-adrenoceptors in the human atrioventricular conducting system: a comparison of patients with idiopathic dilated cardiomyopathy and ischemic heart disease.

The density and distribution of beta 1- and beta 2-adrenoceptors in the atrioventricular conducting system and interatrial and interventricular septa from human hearts with idiopathic dilated cardiomyopathy and ischemic heart disease was determined by quantitative autoradiography using (-)[125I]cyanopindolol and the selective beta 1-adrenoceptor antagonist CGP 20712A and the selective beta 2-adrenoceptor antagonist ICI 118,551. Both beta 1- and beta 2-adrenoceptors were present in the atrioventricular node, bundle of His, interatrial and interventricular septa. No differences in the density or proportions of beta 1- and beta 2-adrenoceptors in the atrioventricular node, bundle of His, interatrial septum and interventricular septum were observed between hearts with idiopathic dilated cardiomyopathy or ischemic heart disease (P > 0.05) so further analysis did not distinguish between the two aetiologies. The density of beta 1-adrenoceptors was lower in the bundle of His (5.0 +/- 1.7 fmol/mg protein) than in the atrioventricular node (22.2 +/- 5.7 fmol/mg protein, P < 0.05), the interatrial septum (29.6 +/- 4.5 fmol/mg protein, P < 0.001) and interventricular septum (24.9 +/- 5.2 fmol/mg protein, P < 0.005, n = 8 for all values). The atrioventricular node, interatrial and interventricular septa had similar densities of beta 1-adrenoceptors (P = 0.60, ANOVA). The distribution of beta 2-adrenoceptors in the atrioventricular node (21.5 +/- 4.1 fmol/mg protein), bundle of His (12.9 +/- 2.6 fmol/mg protein) and atrial (16.7 +/- 2.3 fmol/mg protein) and septal myocardium (13.8 +/- 2.5 fmol/mg protein, n = 8 for all values) was uniform (P = 0.18, ANOVA). The percentage of beta 1- and beta 2-adrenoceptors in the atrioventricular node, bundle of His, interatrial and interventricular septa was uneven (P < 0.001, ANOVA). There was a higher proportion of beta 2-adrenoceptors in the bundle of His (72 +/- 6%) than in the atrioventricular node (51 +/- 3%, P < 0.01), interatrial septum (36 +/- 1%, P < 0.001) and interventricular septum (36 +/- 1%, P < 0.001).

Effects of prolonged infusion of dopexamine on beta 1- and beta 2-adrenoceptors in guinea-pig myocardium.

1. The effects of 7-day infusion of dopexamine (50 and 200 micrograms kg-1h-1) were examined on beta 1- and beta 2-adrenoceptors in guinea-pig left ventricular membranes. 2. Receptor binding performed using the high affinity radioligand (-)-[125I]-cyanopindolol (CYP) and the beta 1-adrenoceptor antagonist CGP 20712A showed that treatment with dopexamine 200 micrograms kg-1h-1 caused a 45% reduction in beta 2-adrenoceptors and a small but not significant increase in beta 1-adrenoceptors. 3. Functional effects of dopexamine were examined in the guinea-pig isolated electrically driven left atria and K(+)-depolarized uterus. Dopexamine was an antagonist at beta 1-adrenoceptors in left atria (pKB = 4.49), and a partial agonist at beta 2-adrenoceptors in the uterus (alpha = 0.78, pD2 = 6.59). 4. The effects of dopexamine on beta 1- and beta 2-adrenoceptor density in guinea-pig ventricular myocardial membranes may be related to agonist activity at beta 2-adrenoceptors and antagonist activity at beta 1-adrenoceptors.

Coexistence and localization of beta 1- and beta 2-adrenoceptors in the human heart.

Quantitative autoradiography was used to determine the location and density of beta 1- and beta 2-adrenoceptors in the right atrium (RA), left ventricular free wall (LVFW), right ventricular free wall (RVFW), interventricular septum (IVS), right atrium from an area near the atrioventricular node (RAAV) and cardiac nerves (N) taken from a patient with end-stage cardiac failure. The densities of beta-adrenoceptors detected by the non-selective beta-adrenoceptor antagonist radioligand (-)-[125I] cyanopindolol (50pM) were 4.93 (N), 10.6 (RVFW), 12.2 (RA), 12.4 (IVS), 15.8 (LVFW) and 18.7 fmol (mg protein)-1 (RAAV). The proportion of beta 2-adrenoceptors ranged from 19.5% (RAAV) to 95% (N). RA taken from patients with ischaemic heart disease had a higher density of beta-adrenoceptors (29.3 fmol (mg protein)-1). The results suggest that both beta 1- and beta 2-adrenoceptors are down-regulated in patients with end-stage cardiac failure. Positive inotropic responses were established to (-)-isoprenaline, RO363 (beta 1-selective), procaterol (beta 2-selective) and dopexamine in the absence or presence of the antagonist CGP 20712A (beta 1-selective) or ICI 118,551 (beta 2-selective) in electrically driven human right atrial appendage strips. RO363 and procaterol were nearly full agonists in this preparation and produced their responses through activation of beta 1- or beta 2-adrenoceptors, while dopexamine was a partial agonist which produced its inotropic responses through activation of both receptor subtypes. These studies demonstrate the presence and location of beta 1- and beta 2-adrenoceptors in the human heart.