Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes.

Signal transmission by insulin involves tyrosine phosphorylation of a major insulin receptor substrate (IRS-1) and exchange of Ras-bound guanosine diphosphate for guanosine triphosphate. Proteins containing Src homology 2 and 3 (SH2 and SH3) domains, such as the p85 regulatory subunit of phosphatidylinositol-3 kinase and growth factor receptor-bound protein 2 (GRB2), bind tyrosine phosphate sites on IRS-1 through their SH2 regions. Such complexes in COS cells were found to contain the heterologously expressed putative guanine nucleotide exchange factor encoded by the Drosophila son of sevenless gene (dSos). Thus, GRB2, p85, or other proteins with SH2-SH3 adapter sequences may link Sos proteins to IRS-1 signaling complexes as part of the mechanism by which insulin activates Ras.

Constitutive phosphorylation of the receptor for insulinlike growth factor I in cells transformed by the src oncogene.

Many oncogene products have been shown to bear strong homology to or to interact with components of normal cellular signal transduction. We have previously shown that a glycoprotein band of 95 kilodaltons (kDa) becomes tyrosine phosphorylated in chick cells transformed by Rous sarcoma virus and that tyrosine phosphorylation of this protein band correlates tightly with phenotypic transformation in cells infected with a large and diverse panel of src mutants (L. M. Kozma, A. B. Reynolds, and M. J. Weber, Mol. Cell. Biol. 10:837-841, 1990). In this communication, we report that a component of the 95-kDa glycoprotein band is related or identical to the 95-kDa beta subunit of the receptor for insulinlike growth factor I (IGF-I). We found that the beta subunit of the IGF-I receptor comigrated on polyacrylamide gels with a component of the 95-kDa glycoprotein region from src-transformed cells under both reducing and nonreducing gel conditions and had a very similar partial phosphopeptide map. To further test the hypothesis that the beta subunit of the IGF-I receptor becomes tyrosine phosphorylated in cells transformed by pp60src, a human cell line that expressed the IGF-I receptor was transformed by src. Comparison of IGF-I receptors immunoprecipitated from normal and transformed cells revealed that the beta subunit of the IGF-I receptor became constitutively tyrosine phosphorylated in src-transformed cells. Moreover, IGF-I receptor phosphorylation induced by src was synergistic with that induced by the hormone: IGF-I-stimulated autophosphorylation of the receptor was much greater in src-transformed cells than in untransformed HOS cells even at maximal concentrations of IGF-I. This increased responsiveness to IGF-I was not due to increases in receptor number, time course of phosphorylation, or affinity for hormone. Finally, no IGF-I-like activity could be detected in culture supernatants collected from the src-transformed cells, suggesting that the increased receptor phosphorylation observed in the src-transformed cells may be mediated by an intracellular mechanism rather than an external autocrine stimulation. Our data demonstrate that the IGF-I receptor becomes constitutively tyrosine phosphorylated in src-transformed cells. This finding raises the possibility that pp60v-src alters growth regulation at least in part by phosphorylating and activating this growth factor receptor.

Glycoprotein tyrosine phosphorylation in Rous sarcoma virus-transformed chicken embryo fibroblasts.

The level of tyrosine phosphorylation of cellular glycoproteins isolated by wheat germ agglutinin chromatography in cells infected with a variety of kinase-positive/transformation-defective src mutants was examined in an effort to identify cellular membrane proteins whose phosphorylation correlates with phenotypic transformation. We have identified two glycoproteins, with molecular masses of 95 and 135 kilodaltons, whose phosphorylation correlates with morphological transformation, growth in soft agar, and an increase in the rate of 2-deoxyglucose uptake. The strong correlation obtained between transformation and phosphorylation of these proteins suggests that they may be substrates for pp60src which are important in the process of transformation.

The genetics of Graves' disease: HLA and disease susceptibility.

To relate genetic variation in Graves' disease (GD) susceptibility to polymorphism at MHC loci, clinical and family studies were undertaken in eastern Hungary. Among 1980 relatives of 534 index patients, 2.9% of siblings, 2.7% of offspring, and 3.0% of parents had GD. HLA haplotype combinations in affected sibling pairs were determined in the present data and combined with data in the literature (12 sibling pairs from Farid 1981, 12 from Chan et al. 1980, and 15 from Sasazuki et al. 1983); 43, 23, and 1 affected sibling pairs shared, respectively, 2, 1, and 0 HLA haplotypes. This distribution is inconsistent with simple dominant inheritance, but is consistent with simple recessive inheritance of HLA-related susceptibility over a range of gene frequencies (0.2-0.4). A frequency of 0.3 gives the best fit and is consistent with penetrance of 7.1% for the recessive susceptibility genotype; the data, however, can accommodate penetrance values up to 16%. The distribution of HLA haplotypes in 33 families related disease susceptibility more strongly to DR than to other loci. The distribution of HLA-B8 genotypes in 256 patients was in close agreement with Hardy-Weinberg equilibrium proportions, also favoring recessive inheritance of MHC-related susceptibility. The probability that an individual will be affected with GD can be predicted, based on sex, HLA genotype, and family history. For example, 14.9% of DR3-positive women with an affected first degree relative are likely to be affected. These predictions can be tested as family data accumulate.

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.

Studies on acute myelomonocytic leukemia in LBF1 rats.

Granulocytic leukemia was induced in Long-Evans (LE) rats by using the Huggins and Sugiyama method. After serial passage the cells became transformed. The newly transformed cells could be transplanted to LBF1 hybrid rats and observed more readily. A quantity of 10(8) cells/100 g body weight was injected intravenously and after 2-3 weeks myelomonocytic leukemia developed. By examining the bone marrow, spleen and lymph nodes, cytochemical tests verified this transformation. Transplanting 10(2)-10(4) cells under the renal capsule, a quickly growing solid tumor was observed, which caused metastasis to the parathymical lymph nodes and peritoneum. The investigation of oncogene expression for the myc and ras families revealed the presence of myc p62 and ras p21 oncoproteins in the tumor cells by using monoclonal antibodies in immunohistochemical tests. LBF1 rats proved to be good models in obtaining solid tumor growth and myelomonocytic leukemias, equivalent to human M4-M5 type leukemia.

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.

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.

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.

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.

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.

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.

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.

[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.

[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.