Copy number of cancer genes predict tumor grade and survival of pancreatic cancer patients.

Pancreatic cancer is on the increase. While means of early diagnosis are being sought, it continues to present late. Prognostication is based on patient and tumor characteristics, including expression or mutation of cancer-related genes. Few studies have examined the impact of the amplification of these genes on the outcome of pancreatic cancer. We have now used a non-radioisotopic slot-blot technique to relate gene copy numbers of p53, c-myc and K-ras to tumor grade and survival. Outcomes were corrected for patient characteristics, tumor location and TNM staging. The Kaplan-Meier test for likelihood of survival showed that increase in copy number of the two oncogenes and loss of p53 were associated with non-significant reduction in survival. When these variations in cancer gene copy numbers were, however, examined by logistic regression analysis in the context of patient and tumor characteristics, survival was negatively related to K-ras amplification (p = 0.0291). Tumor grade, but not survival was positively related to loss of p53 gene copy (p = 0.0131) as well as c-myc amplification (p = 0.0248). Thus using a simple non-radioisotopic technique for the detection of cancer gene copy number in association with patients and disease characteristics, we could predict survival on the one hand and tumor behavior on the other. Such information could be used to plan initial and follow-up therapy.

C-myc amplification and cluster analysis in human gastric carcinoma.

The tumour samples ot 23 patients (9 male, 14 female, aged 28-85) were randomly selected for the study. DNA was isolated from paraffin embedded tissue for quantitative dot-blot hybridization, in order to determine the amplification values for the c-myc and K-ras oncogenes. The clinical and histological parameters studied were as follows: grade, TNM staging system, Lauren's histological type, localization and the severity of the disease. Amplified c-myc was found in 6 cases. Amplification was concomitant with c-myc overexpression detected with immunohistochemical staining. The amplification--9.1-fold on the average (ranging from 2.12 to 18.2) was significantly associated with the presence of distant metastasis (corr. coeff.: 0.5623, p < 0.01), but with none of the other parameters. No case with K-ras amplification was recorded. The result of the multivariate cluster analysis proved that age was the decisive factor in the segregation process. This age-related distribution (69 vs. 40, p < 0.001), however, did not coincide with either the incidence of distant metastasis or c-myc amplification.

[Investigation of oncogene amplification or deletion, and oncoprotein expression in papillary thyroid cancer] / Onkogén-amplifikáció, illetve -deléció és onkoprotein-expresszió immunhisztokémiai vizsgálata papilláris pajzsmirigyrákban.

AIM: Assessment of occurrence and possible prognostic significance of c-myc and Ha-ras amplification, p53 deletion and overexpression of cyclin D1, p53 and p21 in papillary thyroid cancer. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded tumor tissue from 24 patients were investigated. Dot-blot DNA hybridization was used to detect oncogene amplification or deletion. The expression of oncoproteins was determined by immunohistochemical method. RESULTS: In our samples neither Ha-ras amplification nor p53 deletion were found. Low c-myc amplification (mean: 2.55) occured in 4 cases (17%). p53 protein was detected in 16 samples (66.6%), with p21 expression (chi(2)=7.02, p<0.01) in 6 cases (25%). The p53 expression did not influence the tumor fenotype. Cyclin D1 overexpression was found in 12 cases (50%), it was often associated with p21 expression (chi2=10.1, p<0.001) and in inverse relation to the tumor lymphocytic infiltration (chi(2)=5.35, p<0.05). Increased expression of estrogen receptor was shown in 4 cyclin D1 positive samples (17%). CONCLUSIONS: The p53 detected in our study is likely not to be mutant protein in all cases because its presence was associated with p21 expression that the mutant protein cannot induce and also it did not mean more aggressive tumor phenotype. The connection of cyclin D1 overexpression with the lymphocytic infiltration of the tumor suggests that the increased expression of cyclin D1 means poor prognosis. The coexpression of cyclin D1 and p21 raises the modulative character of the p21 protein, thought to be a tumor suppressor originally, but we find a CDK-independent, estrogen receptor mediated effect of cyclin D1 more likely, which has been described in breast cancer and is also proved by the coexpression of cyclin D1 and estrogen receptor detected here.

[Angioneurotic edema induced by angiotensin converting enzyme inhibitors]. / Angiotenzinkonvertáló enzim inhibitorok okozta Quincke-oedema.

Angioneurotic oedema is one of rare side effects of angiotensin converting enzyme inhibitors, its incidence is around 0.1-0.2%. Angio-oedema most commonly develops in the first 4 weeks of the treatment, but it can be observed later, after several months or even years. The association between the oedema and the drug intake can be difficult to recognize if the oedema is of delayed type and because the attacks can disappear spontaneously without discontinuation of the drug. The angioneurotic oedema is tend to be worsening during the treatment, and finally the obstruction of the upper respiratory tract can be fatal. The affected sites are the face, lips, tongue, upper respiratory tract, and the oedema can also develop in the gastrointestinal tract with abdominal pain and diarrhea, which can be misdiagnosed. The pathomechanism is thought to be rather biochemical than immunological. The pathogenetic factors are under investigation nowadays, but the increased level of bradykinin seems to be the most important factor. Authors treated 248 patients with angioneurotic oedema in the Department of Dermatology (Semmelweis Hospital, Miskolc) between January of 1997 and December of 2000, 44 patients took angiotensin converting enzyme inhibitors, and 16 patients were suspected as suffering from angio-oedema induced by this drug. All of the patients remained symptom-free after the adequate treatment and discontinuation of the suspected drug. Authors describe the clinical picture of the angio-oedema, the risk factors, and the contraindications of the angiotensin converting enzyme inhibitor treatment.

Diffraction of x rays in capillary optics.

Propagation of x rays generated by a small-diameter incoherent source through the capillary waveguide that satisfies the multimode condition is studied with the Fresnel-Kirchhoff diffraction theory. The strong influence of diffraction on the propagation is demonstrated. The diffraction phenomenon is manifested by the appearance of diffraction fringes in both the guide channel and the far-field zone of the capillary output. Experimental data that confirm such behavior of the x-ray radiation is also presented. The results confirm the interference effects recently observed in some experiments on the grazing reflections of x rays in single- and multiple-capillary optics.

Possible role of normal development in carcinogenesis.

Apart from intercellular communication and co-operation, the selection pressure on the initiated cells to give rise to a malignant clone seems to depend on the developmental status of the target organ as well as the growing capacity of the prospective tumour. According to our theoretical approach, slowly growing tumours are counter selected and unable to survive in rapidly growing normal tissue.

Age-dependent variation of doubling times in malignant disorders: why are the doubling times of tumours in childhood shorter than in adulthood?

The proportion of patients with any given type of cancer in relation to all cases with malignant disorders in the same age-group exhibits a characteristic age-dependent variation. The values of age of maximal relative frequency (AMRF) were determined from statistics for seven cancer clusters grouped by target organ. The results of this study reveal that there exists a theoretical way of estimating AMRF by the linear combination of the approximative average values of tumour doubling times and the age of half-time development of the respective organ. The good correlation (corr. coeff. = 0.985, P < or = 0.001) between the observed and calculated values for AMRF makes the standard error of the calculation as low as 7.3 years. The conclusion is that in young developing organisms, only those tumours with short doubling time are likely to exist and survive, whereas later, during the period of organic involution and weakening cell-cell cooperation, more and more cancer types of longer doubling time can establish themselves. It seems that weak cellular cooperation yields way to malignancy; nevertheless, the normal growth rate of the target tissue has to be exceeded by the potential tumour. A slowly growing tumour in rapidly growing normal tissue is counterselected.

The amino terminus of insulin-responsive aminopeptidase causes Glut4 translocation in 3T3-L1 adipocytes.

The insulin-responsive aminopeptidase (IRAP) is a constituent of the vesicles that contain the insulin-regulated glucose transporter (Glut4). Like Glut4, IRAP translocates to the cell surface in response to insulin. Microinjection into 3T3-L1 adipocytes of a glutathione S-transferase (GST) fusion protein containing the cytosolic portion of IRAP (GST-IRAP-(1-109)), resulted in translocation of Glut4 to the cell surface. Immunostaining of 3T3-L1 adipocytes for Glut4 showed that the percentage of cells with substantial cell surface Glut4 was 10% in unstimulated cells, 8% following injection of GST, and 27% following injection of GST-IRAP-(1-109). Increased cell surface Glut4 occurred within 5-10 min following injection and was maintained for at least 4 h. A fusion protein containing only 28 amino acids from IRAP (GST-IRAP-(55-82)) was as effective in increasing cell surface Glut4 as stimulation with 100 nM insulin (44% versus 43%, respectively). In contrast to insulin-stimulated Glut4 translocation, the redistribution of Glut4 following injection of GST-IRAP-(55-82) was not blocked by wortmannin or co-injection with a SH2 domain from the regulatory subunit of phosphatidylinositol 3-kinase. These data suggest that the amino terminus of IRAP interacts with a retention/sorting protein that also regulates the distribution of Glut4 in insulin-responsive cells.

Spectrofluorimetric determination of diethazine and promethazine in pharmaceutical preparations.

A simple, sensitive and accurate spectrofluorimetric method is described for the quantitative determination of diethazine and promethazine either in the pure form or in its pharmaceuticals. The method is based on the formation of red fluorescent product of these drugs with Au(III). A linear calibration graph is obtained over the range 0.05-100 p.p.m. of diethazine and promethazine.

Investigation of c-myc and K-ras amplification in renal clear cell adenocarcinoma.

Tumour DNA samples isolated from 36 patients with renal clear cell carcinoma were investigated for c-myc and K-ras amplification, using a quantitative dot-blot hybridization. The characteristic clinical and histological parameters involved in the statistical analysis were age, sex, histological grade of the tumour, the TNM staging system, tumour size and weight, vascular invasion and the quality of life. The goal of the study was to estimate the prevalence as well as the prognostic value of the amplification of the oncogenes in question. Amplified c-myc (2.47-fold on the average) was found in three specimens (8.3%), showing slight correlation with intravasation (P > 0.05, n.s.). K-ras amplification (2.93-fold) detected in six tumours (16.6%) was shown to significantly correlate with both histological grade (2.2 vs. 1.8, P < 0.05) and tumour size (15 vs. 8 cm, P < 0.05). In cases with amplified K-ras also lymph node involvement was somewhat more frequent (P > 0.05, n.s.). No coamplification of these oncogenes was observed. The results of the study suggest that K-ras amplification may account for a more rapid progression of the disease.

Is the presence of distant metastasis associated with c-myc amplification in gastric cancer?

The expression of the c-myc oncogenes has already been reported in human gastric carcinoma. Overexpression can be the consequence of oncogene amplification and often correlates with different prognostic factors. Authors investigated the value of c-myc oncogene amplification in 23 patients (9 male, 14 female, aged 28-85 yrs) with gastric cancer and its correlation to the following clinical and histopathological parameters: grade, TNM stage, Lauren's type, localisation and severity of disease. DNA was isolated from formalin-fixed, paraffin embedded tissue for quantitative dot-blot hybridisation. Amplified c-myc was found in 6 out of 23 cases. Its values ranged from 2.12 up to 18.2 (average 9.1). Significant association was found between the presence of c-myc amplification and distant metastasis (corr. coeff.: 0.5623, p < 0.01). High scores of the other parameters also correlated with c-myc, albeit not significantly. The result of cluster analysis, based on the similarity of the parameter values for the individual patients proved that the age was the decisive factor in creating two groups. The distribution of patients into these groups did not seem to coincide with the presence of c-myc amplification or distant metastasis, inspite of the proved correlation between them.

Time-resolved line shape studies of Nd : YAG laser-induced microplasmas arising from gold surfaces.

A systematic study of the time evolution of the line shape of radiation emitted by a gold plasma and an exact line intensity calculation was carried out. The emission of the hot and dense plasma produced by a Q-switched Nd:YAG laser in atmospheric air was measured by a time-gated optical multichannel analyzer. Asymmetric Lorentz-type profile equations were tested for two gold lines (406.51, 389.79 nm) as a function of time. A strong broadening, asymmetry and shift is observable up to 800-1000 ns after the laser pulse. Spectral profiles of the delayed (with 0.8-1.0 micros) and time-integrated (gate time of 2.5 micros) measurements were found to be well represented by a symmetric Lorentz-type curve.

Is cancer promotion associated with dysfunction in co-operation between differentiated cells?

In a selection-based computer model system we demonstrated that deteriorating cellular co-operation between differentiated cells could result in positive selection for initiated cells of high proliferative capacity. The ratio of the initiated cells to their normal counterparts increased from 0.47 to 0.63 when the strength of co-operation decreased to one hundredth. The correlation proved to be significant. A number of bioactive substances involved in cell-to-cell communication are already registered as cocarcinogens. Whether this approach can explain the role of other promoting agents remains to be answered. It is also possible that the high incidence rate of old-age cancer may be in part accounted for by this kind of co-operational failure during senescence.

Induction of renin release from isolated glomeruli by inorganic mercury(II).

Mercury(II) ions are known to accumulate in the kidney and their effect upon the renin-angiotensin system has also been described. The question, however, whether mercury(II) also exerts direct effect on the juxtaglomerular cells (JGC) to induce renin release remained to be answered. Suspension of isolated glomeruli was used to measure the mercury(II)-induced renin release in vitro. The glomeruli were isolated from female BALBc mice. HgCl2 was found to be capable of inducing renin release directly from JGC. The effect is concentration-dependent (P < 0.05, r = 0.914 and P < 0.01, r = 0.982, with and without Neutral Red vital staining) and becomes apparent already at a mercury(II) ion concentration as low as 1 microM. The renin-releasing effect of the mercury ion is to be inhibited by dithiothreitol (DTT) (renin activity 20.37 vs. 2.60 ng/ml.h in supernatant) as well as the elevated osmotic concentration of the incubating bath medium (20.37 vs. 6.84 ng/ml.h). This suggests that certain membrane sulfhydryl groups are implicated in the process on the one hand, and it is also in accordance with the known sensitivity of the renin granules to osmotic pressure on the other hand. Light and electron micrographs also demonstrate the direct, effective role of Hg(II) in the renin release process. Therefore, it is assumed that apart from its influence on tubulo-glomerular feedback a direct way of action of mercury(II) on renin release must also be taken into account.

Functional roles for the pleckstrin and Dbl homology regions in the Ras exchange factor Son-of-sevenless.

Activation of p21ras by receptor tyrosine kinases is thought to result from recruitment of guanine nucleotide exchange factors such as Son-of-sevenless (Sos) to plasma membrane receptor substrates via adaptor proteins such as Grb2. This hypothesis was tested in the present studies by evaluating the ability of truncation and deletion mutants of Drosophila (d)Sos to enhance [32P]GTP loading of p21ras when expressed in 32P-labeled COS or 293 cells. The dSos catalytic domain (residues 758-1125), expressed without the dSos NH2-terminal (residues 1-757) or adaptor-binding COOH-terminal (residues 1126-1596) regions, exhibits intrinsic exchange activity as evidenced by its rescue of mutant Saccharomyces cerevisiae deficient in endogenous GTP/GDP exchange activity. Here we show that this dSos catalytic domain fails to affect GTP p21ras levels when expressed in cultured mammalian cells unless the NH2-terminal domain is also present. Surprisingly, the COOH-terminal, adaptor binding domain of dSos was not sufficient to confer p21ras exchange activity to the Sos catalytic domain in these cells in the absence of the NH2-terminal domain. This function of promoting catalytic domain activity could be localized by mutational analysis to the pleckstrin and Dbl homology sequences located just NH2-terminal to the catalytic domain. The results demonstrate a functional role for these pleckstrin and Dbl domains within the dSos protein, and suggest the presence of unidentified cellular elements that interact with these domains and participate in the regulation of p21ras.

Regulation by insulin of phosphatidylinositol 3'-kinase bound to alpha- and beta-isoforms of p85 regulatory subunit.

The roles of the alpha- and beta-isoforms of phosphatidylinositol (PI) 3'-kinase p85 regulatory subunit were studied with isoform-specific antisera in three model systems in which the insulin receptor mediates rapid phosphorylation of insulin receptor substrate-1 (IRS-1). Insulin receptor signaling stimulated the association of IRS-1 with p85 alpha protein, and p85 alpha-associated PI 3-kinase activity in 3T3-L1 adipocytes, and in transfected Chinese hamster ovary cells (CHO-T) and COS-1 cells expressing high levels of human insulin receptors. While not detectable in 3T3-L1 adipocytes, the p85 beta isoform was also found to associate with IRS-1 in response to insulin receptor activation in COS-1 and CHO-T cells. However, selective immunoprecipitation of p85 beta from unstimulated COS-1 or CHO-T cell lysates was accompanied by higher levels of PI 3-kinase activity than that associated with p85 alpha. Remarkably, the large stimulation of PI 3-kinase activity associated with p85 alpha (7.8 +/- 2.0-fold, n = 6) in insulin-treated CHO-T cells was not observed in p85 beta immunoprecipitates (1.8 +/- 0.6-fold, n = 6), and in COS-1 cells p85 beta-associated PI 3-kinase activity was completely insensitive to stimulation by the insulin receptor. These data suggest the novel hypothesis that binding of p85 beta to IRS-1 complexes in COS-1 and CHO-T cells does not mediate marked activation of PI 3-kinase activity as does p85 alpha.

Investigation of c-myc oncogene amplification in colorectal cancer.

Tumour DNA samples of 20 patients with colorectal carcinoma were tested for c-myc amplification, using a quantitative dot-blot hybridization. Statistical analysis involving clinical and histological parameters like degree of differentiation, Dukes' stage, TNM staging system, age, sex and severity of disease, was applied to estimate the prognostic value of c-myc amplification. The amplification of the investigated oncogene--1.61-fold on the average--was found to significantly correlate with the presence of distant metastasis (corr. coeff.: 0.506, P < 0.05) and the severe course of the disease (corr. coeff.: 0.468, P < 0.05). This result supports the hypothesis that tumour cells with c-myc amplification represent a more malignant and aggressive phenotype. It is also worth noting that both c-myc amplification and formation of distant metastasis are late events in the progression of colorectal cancer, which accounts for the more severe course of the disease.