Functional categories of TP53 mutation in colorectal cancer: results of an International Collaborative Study.

BACKGROUND: Loss of TP53 function through gene mutation is a critical event in the development and progression of many tumour types including colorectal cancer (CRC). In vitro studies have found considerable heterogeneity amongst different TP53 mutants in terms of their transactivating abilities. The aim of this work was to evaluate whether TP53 mutations classified as functionally inactive (< or=20% of wildtype transactivation ability) had different prognostic and predictive values in CRC compared with mutations that retained significant activity. MATERIALS AND METHODS: TP53 mutations within a large, international database of CRC (n = 3583) were classified according to functional status for transactivation. RESULTS: Inactive TP53 mutations were found in 29% of all CRCs and were more frequent in rectal (32%) than proximal colon (22%) tumours (P < 0.001). Higher frequencies of inactive TP53 mutations were also seen in advanced stage tumours (P = 0.0003) and in tumours with the poor prognostic features of vascular (P = 0.006) and lymphatic invasion (P = 0.002). Inactive TP53 mutations were associated with significantly worse outcome only in patients with Dukes' stage D tumours (RR = 1.71, 95%CI 1.25-2.33, P < 0.001). Patients with Dukes' C stage tumours appeared to gain a survival benefit from 5-fluorouracil-based chemotherapy regardless of TP53 functional status for transactivation ability. CONCLUSIONS: Mutations that inactivate the transactivational ability of TP53 are more frequent in advanced CRC and are associated with worse prognosis in this stage of disease.

Multitemperature single-strand conformation polymorphism.

Changes of gel temperature during single-strand conformation polymorphism (SSCP) electrophoresis increase the sensitivity of mutation detection in polymerase chain reaction (PCR) products and significantly reduce the overall time and costs of analysis. Based on these findings, a new method for single nucleotide polymorphism (SNP) and point mutation detection--multitemperature single-strand conformation polymorphism (MSSCP) was devised. In order to control the gel temperature with 0.1 degrees C accuracy during electrophoresis, new equipment was developed. We demonstrated that increasing the gel temperature by 8 degrees C or decreasing it by 10 degrees C from 23 degrees C led to the disappearance of all electrophoretic differences between five alleles of exon 8 of the human p53 gene during the SSCP analysis. The interesting result was the detection of two additional SNPs (out of seven analyzed) in exon 7 of the human PAH gene during a one hour MSSCP electrophoresis. This result is better than that obtained by three classical SSCP analyses of the same samples at different but constant gel temperatures. We advocate the MSSCP technology as a fast, reliable, and cost-effective tool for the screening and preselection stage of genomics surveys, especially when a high variability of the analyzed DNA fragment is expected.

Mutations, methylation and expression of CDKN2a/p16 gene in colorectal cancer and normal colonic mucosa.

The status of CDKN2a gene, coding for p16 and p19ARF proteins, was examined in 55 colorectal cancers. Polymerase chain reaction (PCR), single stranded conformational polymorphism and sequencing revealed 1 case of CDKN2a mutation. Methylation-specific PCR detected p16 locus methylation in 37 (73%) of 51 normal samples and 29 (53%) of 55 cancers (P=0.035). p16 transcript absence (assessed by reverse transcription-polymerase chain reaction) was noted in 10 (45%) of 22 normal samples and four (14%) of 29 cancers (P=0.012) and correlated with gene methylation (P=0.036). The decreasing frequency of p16 silencing in cancer comparing to normal mucosa does not support the postulated role of p16 in colorectal carcinogenesis.

Increased activity of MAP, p70S6 and p90rs kinases is associated with AP-1 activation in spontaneous liver tumours, but not in adjacent tissue in mice.

Growth factor-responsive protein kinases regulate expression of genes involved in cell cycle control, cell proliferation and differentiation. To better understand the role of these kinases in the abnormal proliferation of malignant cells, we examined basal and epidermal growth factor (EGF)-inducible mitogen-activated protein kinase (MAPK), p70S6k and p90rsk activities in spontaneous hepatocellular neoplasms (adenomas and carcinomas) from CBA-T6 mice and in L1 sarcoma tumours implanted in livers of BALB/c mice. In spontaneous and implanted hepatic tumours, basal cytoplasmic and nuclear MAPK, p70S6k and p90rsk activities were significantly higher compared to the activities found in the part of the liver uninvolved by the tumour. Interestingly, the activities of these enzymes in the uninvolved tissue of the livers harbouring the tumour were higher compared to the livers from control mice. Basal kinase activities correlated with tumour morphology; they were lower in adenomas than in carcinomas and sarcomas. In contrast to basal activities, EGF-triggered kinase responses in normal livers and hepatic tumours were indistinguishable. Activating protein-1 (AP-1) DNA-binding activity was detected in tumours but not in the adjacent tissues. Constitutively activated kinases and AP-1 transcription factor found in hepatic malignancies are reminiscent of cells activated by EGF, suggesting that EGF and its intracellular effectors play a role in these malignancies.

In search of immunogenic Helicobacter pylori proteins by screening of expression library.

BACKGROUND: Prevention of Helicobacter pylori infection may help to control related gastritis, peptic ulcer and cancer. Of the possible preventive measures, immunization was successfully employed in various animal studies. However, no immunization protocol has been accepted for humans. A better characterization of the immune response against the pathogen may be required before a human vaccine is developed. AIM: To identify bacterial proteins which induce an immune response in infected humans or H. pylori-immunized rabbits. METHODS: An expression library of H. pylori genes was screened with sera from infected humans and from immunized rabbits. Positive clones were partially sequenced and identified on the basis of a homology search of a H. pylori genome database. Encoded proteins were expressed directly from positive clones and analyzed by SDS-PAGE/Western blot techniques. RESULTS: 114 positive clones were isolated: 79 by screening with human sera and 35 by screening with rabbit sera. Western blot analysis demonstrated that selected clones encoded one or more strongly immunoreactive proteins. 64 clones selected with human sera had no counterparts among clones from screening with rabbit serum. 13 of these clones encoded a total of 21 unknown H. pylori proteins. 17 clones selected with rabbit sera were not immunostained with human sera. They represent 2 various regions of the H. pylori genome which encoded 3 bacterial proteins of unknown function. CONCLUSIONS: Screening of H. pylori expression library identified immunogenic proteins - potential vaccine antigens.

VacA genotyping directly from gastric biopsy specimens and estimation of mixed Helicobacter pylori infections in patients with duodenal ulcer and gastritis.

BACKGROUND: The vacA genotypes and the cagA gene status were investigated in 80 Helicobacter pylori-infected patients with duodenal ulcer (DU) and 49 with gastritis only. METHODS: Lysates of gastric biopsy specimens were used directly for polymerase chain reaction-based detection. RESULTS: The ml subtype was found in 36% and 31% and the m2 in 36% and 46% of specimens from patients with DU and gastritis, respectively (P > 0.05). In 15% of samples the midregion remained unclassified. The prevalence rate of s1 subtypes was higher in cases of DU (69%) than in gastritis (43%) (P < 0.0001); the opposite correlation was observed for s2. The cagA gene was detected in 80% of patients with DU and in 52% of those with gastritis (P < 0.0001). Infections with multiple H. pylori strains exceeded 50% in both groups. CONCLUSIONS: These results suggest that vacA s1 genotype and cagA+ status are associated with higher DU prevalence and that mixed H. pylori infections are very common in our geographic region.

Increased constitutive activity of mitogen-activated protein kinase and renaturable 85 kDa kinase in human-colorectal cancer.

Protein kinases play a key role in intracellular signalling, participating at multiple levels along the transduction cascades that trigger mitogenic response. Because protein kinases are involved in mitogenic pathways, they are likely to play a role in the abnormal proliferation of malignant cells. In this study we compared activity of mitogen-activated protein (MAP) kinase and several renaturable kinases in homogenates of 30 surgically resected colorectal cancers and their adjacent normal tissues. Using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and membrane autophosphorylation assay on homogenates obtained from normal colon mucosa and adenocarcinoma, we identified at least four renaturable kinases (50, 55, 85, 200 kDa). Compared with adjacent tissue, in most of the cancer samples only the 85-kDa kinase exhibited a higher level of autophosphorylation activity than those in normal matched tissue (P < 0.001). Moreover, the 85-kDa kinase from nearly all cancer homogenates showed faster electrophoretic mobility than the 85-kDa kinase from normal tissue homogenates. Interestingly, the 50-kDa kinase had significantly lower autophosphorylation activity in cancer tissues than those of normal tissue (P< 0.05). To assess p42-p44 MAP kinase activity, proteins were immunoprecipitated from adjacent colon mucosa and adenocarcinoma with anti-extracellular signal-related kinase (ERK) 1/2 antibodies, and MAP kinase activity was measured using MBP as a substrate. These studies revealed that MAP kinase activity in colorectal cancer was significantly higher (P < 0.001) than that in adjacent mucosa. Thus, the constitutive activity of MAP kinase and autophosphorylation activity of 85-kDa kinase are increased, whereas the autophosphorylation activity of another kinase, 50 kDa, is decreased in colorectal adenocarcinoma. However, although signal transduction pathways are markedly altered in this cancer, neither p42/p44 MAP kinase activity nor 85-kDa autokinase activity could be correlated with the established prognostic indicators.

Expression of bcl-2 and bax in TGF-beta 1-induced apoptosis of L1210 leukemic cells.

TGF-beta1 is a multifunctional regulatory peptide (25 kDa) inducing growth arrest and apoptosis in many normal and neoplastic cells. In the present study, the involvement of proapoptotic (bax) and antiapoptotic (bcl-2) genes in the molecular mechanism of TGF-beta1-induced apoptosis of L1210 leukemic cells was investigated. Bax transcript was measured using the RT-PCR method with GAPDH as a "housekeeping gene" control, whereas Bcl-2 protein was determined using flow cytometry (FITC-conjugated monoclonal anti-Bcl-2 antibody and FITC-conjugated mouse anti-IgG1 antibody as a negative control). Apoptosis was evaluated using fluorescence microscopy and flow cytometry after cell staining with DAPI and sulforhodamine or propidium iodide and Hoechst 33342. ROS generation was assessed by flow cytometry using the oxidation-sensitive fluorescent marker C-DCDHF-DA. The response of L1210 leukemic cells to TGF-beta1 was two-directional: 1) partial arrest of the cell cycle at G1-S transition, and 2) induction of apoptotic cell death. TGF-beta1 increased the number of leukemic cells with typical morphological features of apoptosis: cell shrinkage, condensation of chromatin, pyknosis and fragmentation of nuclei, followed by secondary necrosis. DNA cleavage led to a decrease of the nuclear DNA content and the appearance of a hypodiploid peak sub-G1 in the DNA histogram. The extraction of low-molecular weight DNA fragments from apoptotic cells showed that TGF-beta1-induced apoptosis concerned first of all the cells from G1 phase. Two phases of intracellular ROS generation in TGF-beta1-treated cultures were observed: the first (rapid, 60 min after TGF-beta1 administration), and the second (slow, occurring between 24 and 48h of experiment, respectively). The increase of apoptotic cell number in TGF-beta1-treated cultures (2% FCS/RPMI 1640) was associated with the decrease of cell number expressing bcl-2, and with a significant drop of Bcl-2 level in the remaining cells after 24 h. The dose-dependent relationship between TGF-beta1 concentration and Bcl-2 level was nonlinear and described by power series regression. The lowest Bcl-2 level was noted at 1 ng/ml of TGF-beta1 concentration. The increase of Bax mRNA:GAPDH mRNA ratio was observed 3h after TGF-beta1 (1 ng/ml) administration to both the maintenance (2% FCS/RPMI) and growth promoting (10% FCS/RPMI) medium. Regardless of TGF-beta1 treatment, the quantity of Bax transcript was dependent on FCS concentration, being higher in the growth promoting medium. The results of this study indicate that bax may function as a primary response gene and together with lowered Bcl-2 level may determine the induction of apoptotic process in L1210 leukemic cells exposed to TGF-beta1.

The effect of OA on proliferation and polyamine metabolism of K 562 leukemic cells and their responsiveness to natural killer cell activity.

Orotic acid (OA), a known promoter of carcinogenesis, significantly stimulated proliferation of K 562 leukemic cells even at as high a concentration as 0.1 mM. This effect was accompanied by a significant increase of the activity of two key enzymes of the polyamine pathway, i.e. ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC). The induction of ODC activity was associated with increased expression of the ODC gene. The participation of ODC in early events evoked by OA in leukemic cells was confirmed by the decrease of the stimulatory effect of OA on cell proliferation in the presence of alpha-difluoromethylornithine (DFMO)--an irreversible inhibitor of ODC. The involvement of protein kinase C (PK-C) and cyclic nucleotide-dependent kinases in OA action on K 562 leukemic cells was demonstrated by a significant reduction of cell proliferation by addition of H-7 (1-(5-isoquinolinesulphonyl)-2-methylpiperazine). Since PK-C is involved both in induction of ODC activity and in membrane transport of polyamines, H-7 significantly inhibited the proliferation of K 562 leukemic cells even in the presence of OA and exogenous putrescine. The importance of extracellular sources of polyamines for leukemic cell growth was shown by supplementation of the incubation medium with putrescine. Exogenous putrescine significantly enhanced the concentration of spermidine and spermine within the cell and increased the number of cells. The effect of OA on natural killer (NK) cell cytotoxicity was also examined. Rat peripheral blood mononuclear cells were used as effector cells and K 562 cells as targets. OA, progressively with dose, significantly decreased specific lysis when targets were preincubated with it. On the other hand, pretreatment of PBMC effector cells with OA, regardless of the applied concentration, did not affect the amount of 51Cr released from lysed cells. OA as a promoter of carcinogenesis stimulates proliferation of leukemic cells and impairs their responsiveness to NK activity. ODC/polyamine system and PK-C appear to be involved in OA action on K 562 cells. The presented observations are important from a practical point of view, since an elevated blood concentration of OA resulting from the impaired kidney function in hematological proliferative diseases may accelerate their progression.

Effect of orotic acid on TGF-beta 1-induced growth inhibition of L1210 leukemic cells.

UNLABELLED: Transforming growth factor-beta 1 (TGF-beta 1) exerted growth-inhibitory effect on L1210 leukemic cell line, manifested by the decrease in viable and increase in dead cells. The cell death evoked by TGF-beta 1 was both necrotic and apoptotic, quantified by the trypan blue exclusion method and apoptotic index, respectively. The induction of programmed cell death by TGF-beta 1 was confirmed by gel electrophoresis of DNA, where the characteristic 'DNA ladder' resulting from the internucleosomal DNA cleavage was visualized. The enhancement of cell mortality by TGF-beta 1 was associated with the inhibition of ornithine decarboxylase (ODC) expression (measured by the reverse transcriptase-polymerase chain reaction method) and impaired activity of this key enzyme in polyamine synthesis. Orotic acid (OA)--a known tumor promoter--stimulated proliferation of L1210 leukemic cells and diminished the necrotic effect of TGF-beta 1, but it did not change the extent of apoptosis evoked by TGF-beta 1. OA increased the expression of ODC and diminished depressional influence of TGF-beta 1 on transcription and activity of ODC in leukemic cells. IN CONCLUSION: OA is a bioactive compound stimulating the growth of leukemic cells and diminishing the growth-inhibitory effect of TGF-beta 1. ODC gene is probably one of the targets for both OA and TGF-beta 1 influences in L1210 leukemic cells.

Rectal mucosal ornithine decarboxylase activity in familial adenomatous polyposis after ileorectal anastomosis.

Resection of the colon in patients with familial adenomatous polyposis frequently results in the regression of polyps in the remaining rectum, suggesting a reduction of cellular proliferation. These patients remain at risk of developing rectal cancer but whether this risk increases with time is uncertain. Since ornithine decarboxylase activity is associated with cellular proliferation, mucosal ornithine decarboxylase was measured in rectal biopsy specimens from patients with familial adenomatous polyposis after ileorectal anastomosis (n = 36) and from normal controls (n = 30). The relationship between ornithine decarboxylase activity, age, and time from surgery was also examined. Median ornithine decarboxylase activity in familial adenomatous polyposis patients after ileorectal anastomosis (186, interquartile range (IQR) 107-534 pmol/mg protein/h) was not different from that in control subjects (227, IQR 123-374, p = 0.6). When patients were divided into three equal groups according to age, however, younger patients (< 25 years) had significantly higher activity than both older age groups (p < 0.02). Similarly, when patients were stratified according to the time elapsed since surgery, those who had had surgery less than six years previously had a significantly higher ornithine decarboxylase activity than those in whom a longer time interval had elapsed since surgery (p = 0.02). These results indicate that after colon resection, ornithine decarboxylase activity in patients with familial adenomatous polyposis is similar to that in normal controls but seems to fall over time. This may explain the regression of rectal polyps after colonic resection in this disorder.

Induction of ornithine decarboxylase in normal and protein kinase C--depleted human colon carcinoma cells.

To examine the role of protein kinase C (PKC) in induction of human colon adenocarcinoma cell line, DETA/W, by polypeptide growth-promoting factors, ornithine decarboxylase activity (ODC) and DNA synthesis were determined in cells depleted of PKC. PKC depletion was achieved by prolonged cultivation (more than 30 passages) with 10(-6) M phorbol 12-myristate 13-acelate. Lack of PKC in studied cells was proved by measurements of PKC activity and immunoreactivity. Although ODC activities and DNA syntheses in PKC-depleted cells were decreased by about 40-50% compared to normal DETA/W cells, the percentage increase of these mitogen-responsive reactions was quantitatively similar in both cell sublines. These results raise the possibility that not all of the biological responses to growth factors are connected with the activation of calcium-dependent PKC.