Biophenotypes and survival of BRCA1 and TP53 deleted breast cancer in young women.

The aim of this study was to examine the loss of heterozygosity (LOH) of BRCA1 (17q21) and TP53 (17p13.1) in early-onset breast cancer patients; to correlate biopathological characteristics with molecular alterations; and to investigate the survival of LOH-related cancers. BRCA1 and TP53 LOH were evaluated in 78 early-onset breast cancers (< or = 40 years, Group 1) and 80 patients with age > 55 years (Group 2). Cases were characterized for multiple biological markers (ER, PR, proliferation index (PI), NEU and p53). LOH was carried out on microdissected paraffin embedded tissues; microsatellites D17S855 (BRCA1) and D17S786 (TP53) were amplified by fluorescent PCR and analyzed by an automated DNA sequencer. Early-onset breast cancers showed a higher frequency of ductal histotype (89.7% vs. 56.3% p < 0.001), node-positive (53.8% vs. 38.7%), larger size (p = 0.017), higher mitotic rate (p = 0.025), higher nuclear and final grade (p = 0.01 and p = 0.001, respectively). D17S855 LOH was 32.8% in group 1 vs. 21% in group 2; D17S786 LOH was 50.7% vs. 31.3% (p = 0.03), respectively. BRCA1 LOH was correlated with higher PI (p = 0.032) and higher p53 expression (p < 0.001) in group 1 and with higher NEU expression (p = 0.028) in group 2. TP53 LOH was correlated with p53 overexpression (p = 0.03) in group 1. A worse clinical outcome in early-onset LOH related cancers emerged from follow-up data: TP53 and BRCA1 LOH were associated with a shorter relapse free interval (RFI) (p = 0.03) and a poorer overall survival (OS) (p = 0.04), respectively. This study underlines different biological profiles in the two age groups investigated, probably reflecting different mechanisms of carcinogenesis. In accordance with adverse histopathological features in early-onset patients, LOH-related cancers have an unfavorable prognosis.

Sporadic colorectal adenocarcinomas with high-frequency microsatellite instability.

BACKGROUND: Widespread microsatellite instability (MSI) occurs in nearly 15% of sporadic colorectal cancers. Large bowel carcinomas with high-frequency MSI (MSI-H) (instability at > or = 30% of microsatellite loci) are believed to display distinctive pathologic features and to behave less aggressively than microsatellite-stable (MSS) tumors and carcinomas with low-frequency MSI (MSI-L) (instability at < 30% of microsatellite loci). The aim of the current study was to accurately define the clinicopathologic and biologic features of MSI-H sporadic colorectal carcinomas. METHODS: MSI status was evaluated in 216 large bowel adenocarcinomas using polymerase chain reaction (PCR) and 6 microsatellite markers. Tumors that showed instability with at least two microsatellite markers were classified as MSI-H, whereas the other tumors were classified as MSI-L (instability at one locus) or MSS (no instability). Expression of p53, hMLH1, and hMSH2 gene products was determined by immunohistochemistry, and DNA ploidy pattern was determined by flow cytometry. The prognostic significance of MSI status was assessed by univariate and multivariate survival analyses. RESULTS: The significantly different pathologic features of MSI-H carcinomas were proximal location; large size; mucinous and medullary histotype; poor differentiation; expanding pattern of growth; more frequent Crohn-like conspicuous lymphoid reaction; and low incidence of extramural vein invasion. Most MSI-H tumors were DNA diploid (33 of 40 tumors; 82.5%) and p53 negative (34 of 44 tumors; 77.3%). Conversely, DNA aneuploidy and p53 overexpression were observed in 82.3% (130 of 158 tumors; P < 0.0001) and 54.1% (93 of 172 tumors; P = 0.0002) of MSI-L/MSS tumors, respectively. Loss of hMLH1 or hMSH2 expression was detected in a high fraction of MSI-H carcinomas (86. 0%). Patients with MSI-H tumors showed a better clinical outcome than patients with MSI-L/MSS tumors (P = 0.0017). Furthermore, in multivariate analysis that included conventional clinicopathologic parameters, MSI status, and p53 expression as covariates, MSI status was a significant independent prognostic indicator of disease specific survival. CONCLUSIONS: Assessment of MSI status is an essential step in the genetic characterization of large bowel carcinomas and identifies a subset of tumors with distinct clinical, pathologic, and biologic features.

Medullary-type poorly differentiated adenocarcinoma of the large bowel: a distinct clinicopathologic entity characterized by microsatellite instability and improved survival.

PURPOSE: Recent studies suggest the existence of a distinct class of poorly differentiated large bowel adenocarcinomas, usually termed medullary-type adenocarcinomas (MTAs). The aim of the present study was to accurately define the clinical, histopathologic, biologic, and genetic features of this tumor type. MATERIALS AND METHODS: Among 1,265 surgically resected sporadic colorectal carcinomas, 45 MTAs were identified on the basis of the following criteria: predominantly solid growth pattern (at least 70% of the tumor area) and lack of marked nuclear pleomorphism. The clinicopathologic, biologic, and genetic characteristics of MTAs were compared with those of a series of 457 common glandular colorectal adenocarcinomas. RESULTS: The significantly different clinicopathologic features of MTAs were proximal location, large size, invasion into adjacent organs, expanding pattern of growth, low incidence of distant metastases, more frequent conspicuous peritumoral lymphocytic infiltration, and Crohn's-like lymphoid reaction. Furthermore, young patients with MTAs often demonstrated a family history highly suggestive of a hereditary background. Unlike glandular adenocarcinomas, the large majority of MTAs were DNA diploid by flow cytometric analysis (21 of 25, 84%) and p53 negative by immunohistochemistry (36 of 41, 87.8%). In addition, 18 of the 20 MTAs examined by DNA microsatellite analysis demonstrated widespread microsatellite instability (90% of cases). Patients with MTAs showed a better clinical outcome with respect to patients with common poorly differentiated adenocarcinomas (PDAs) (P <.0001) and well- or moderately differentiated adenocarcinomas (WMDAs) (P =.133). In particular, none of the 33 patients with completely resectable stage II and III MTAs developed tumor recurrence during the observation period. Conversely, 24.7% of patients with stage II and III WMDAs and 48.9% of patients with stage II and III PDAs, who had undergone curative surgical resection, died of recurrent disease (P =.01 and P <.0001, respectively). CONCLUSION: All these data strongly indicate that MTAs represent a distinct pathologic entity, with specific histologic appearance and peculiar clinical and genetic features. These tumors need to be classified separately from other poorly differentiated colorectal carcinomas.

Chromosome 18q allelic loss and prognosis in stage II and III colon cancer.

The prognostic significance of chromosome 18q allelic loss was evaluated in a series of 118 patients with curatively resected TNM stage II or stage III colon cancer. Chromosome 18q status was determined on frozen tumour samples, using microsatellite markers and the polymerase chain reaction (PCR). Mean follow-up in surviving patients was 75.9 months. Chromosome 18q allelic loss was significantly related to tumour site, extramural venous invasion, flow cytometric nuclear DNA content and p53 protein expression. Patients whose tumour had no evidence of chromosome 18q allelic loss showed a better disease-free and overall survival than patients whose tumour demonstrated 18q allelic loss. When patients were stratified by tumour stage, a significant survival advantage for patients whose tumour had no allelic loss on chromosome 18q was observed in stage II as well as in stage III disease. In particular, patients with stage II disease whose tumour had no chromosome 18q allelic loss demonstrated an excellent clinical outcome, with a 5-year disease-free survival rate of 96%. In contrast, the 5-year disease-free survival rate of patients with stage II disease and chromosome 18q allelic loss was only 54%. In multivariate analysis, status of chromosome 18q was the only significant independent prognostic factor for both disease-free and overall survival. These results indicate that assessment of chromosome 18q status provides relevant prognostic information in colon cancer and might be employed in the selection of patients for adjuvant therapy.

Human leukemia K562 cells: induction to erythroid differentiation by guanine, guanosine and guanine nucleotides.

BACKGROUND AND OBJECTIVE: Human leukemic K562 cells are able to undergo erythroid differentiation in vitro when cultured with a variety of inducers, leading to increased expression of embryo-fetal globin genes such as the zita, epsilon and gamma-globin genes. Therefore the K562 cell line has been proposed as a very useful in vitro model system for determining the therapeutical potential of new differentiating compounds as well as for studying the molecular mechanism(s) that regulate changes in the expression of embryonic and fetal human globin genes. In this study we explored whether nucleoside triphosphates and related compounds are able to induce differentiation of K562 cells. METHODS: K562 cell differentiation was studied using the benzidine test; hemoglobins were characterized by cellulose acetate gel electrophoresis and mRNA accumulation was investigated by Northern blot analysis. RESULTS: The main conclusion of this paper is that guanine, guanosine and guanine ribonucleotides are effective inducers of K562 cell differentiation. Expression of both Hb Portland and Hb Gower 1 is increased in GTP-induced K562 cells. This increase is associated with greater gamma-globin mRNA accumulation. By contrast, ATP, CTP and UTP are not able to induce erythroid differentiation. INTERPRETATION AND CONCLUSIONS: These findings suggest that guanine, guanosine and guanine ribonucleotides are inducers of erythroid differentiation of K562 cells. This is of some relevance since differentiating compounds have been proposed as antitumor agents. In addition, inducers of erythroid differentiation that stimulate gamma-globin synthesis might be considered in the experimental therapy of hematological diseases associated with a failure in the expression of adult beta-globin genes.

Myelin basic protein inhibits the calcium response to phytohaemagglutinin in human lymphocytes.

Myelin Basic Protein, one of the major membrane protein component of the central nervous system, was used to probe the molecular mechanism of cellular activation by phytohaemagglutinin. Pre-treatment of human lymphocytes with myelin basic protein results in a lower rising of cytosolic concentration of free calcium after stimulation with phytohaemagglutinin. This effect is dependent on myelin basic protein concentration and on the preincubation time of the protein with the cells. It is not due to a interaction between myelin basic protein and phytohaemagglutinin, but appears to be a consequence of the binding of the protein to the cell surface. The reduction of the rise of cytosolic calcium induced by phytohaemagglutinin is specific for the myelin basic protein because other proteins like albumin and protamine have no effect.

Effect of 17 beta-estradiol on calcium response to phytohaemagglutinin in human lymphocytes.

Pre-treatment of human lymphocytes with 17 beta-estradiol diminishes the increase in concentration of cytosolic free calcium after stimulation with phytohaemagglutinin. The effect is dependent on 17 beta-estradiol concentration and on the preincubation time. The effect is not due to an interaction between 17 beta-estradiol and phytohaemagglutinin, but appears to be a consequence of the binding of the hormone to the cell surface. The effect is specific for 17 beta-estradiol, since the alpha isomer and other steroid hormones (progesterone, testosterone, diethylstilbestrol and 5 alpha-androstan) have no effect. Since the effect of the 17 beta-estradiol can be suppressed by treatment of lymphocytes with ouabain, it appears that the effect of estradiol on the rise of cytosolic calcium induced by phytohaemagglutinin is mediated by the (Na, K)-ATPase.

Effect of 17 beta-estradiol on partition behavior of human erythrocytes in dextran-poly(ethylene glycol) aqueous phases.

Preincubation with 1 nM 17 beta-estradiol changes the partition behaviour of human erythrocytes in dextran-poly(ethylene glycol) aqueous phases. Erythrocyte partition decreases reaching a minimum after 5 min of incubation, then slowly increases returning to starting values after 30 min. The effect is specifically induced by the isomer of estradiol, whereas the alpha isomer and other steroid hormones (progesterone, testosterone and 5 alpha-dihydrotestosterone) are uneffective. The effect of the hormone can be suppressed by treatment of erythrocytes with either N-ethyl maleimide, sodium vanadate, or ouabain, or carrying out incubations in potassium-free buffer. These data suggest that the effect of estradiol on phase partition of erythrocytes is mediated by the (Na,K)-ATPase.

A rapid method for purification of myelin basic protein.

A rapid procedure for purification of myelin basic protein has been developed. White matter is delipidated with 2-butanol, and the residue is extracted at pH 7.5 and 8.5. Myelin basic protein is solubilized by extraction in acetate buffer, pH 4.5. The entire procedure requires less than 4 h, and gives homogeneous protein essentially free of protease activity. This procedure can be scaled down to process milligram amounts of white matter; thus it can be very useful for purification of myelin basic protein from very limited amounts of human white matter obtained during surgery.

Half-site reactivity in 6-phosphogluconate dehydrogenase from human erythrocytes.

6-Phosphogluconate dehydrogenase from human erythrocytes was purified by an improved procedure. Binding studies showed that the dimeric enzyme binds 2 mol of NADP+/mol but only 1 mol of NADPH/mol, and that the bindings of oxidized and reduced coenzyme are mutually exclusive. From initial-rate kinetics and inhibition studies, a sequential random-order mechanism is proposed. Double-reciprocal plots with NADP+ as varied substrate show a downward curvature, indicating a negative co-operativity. We suggest that the negative co-operativity observed kinetically is a result of the half-site reactivity for the NADPH. The different binding stoichiometries for NADP+ and NADPH generate a non-linear relationship between the apparent dissociation constant for the NADPH and the concentrations of the NADP+, resulting in a regulatory mechanism highly sensitive to the changes in the NADP+/NADPH ratio.

The NADP binding site on rabbit muscle aldolase.

Rabbit muscle aldolase binds NADPH with a 1:1 stoichiometry and with a dissociation constant 18 microM. Three sites of the dinucleotide are involved in the binding: the adenosyl diphosphate moiety, the nicotinamide-ribose, and the nicotinamide ring. These data show the existence of a specific dinucleotide binding site in the aldolase molecule.

Evidence for the proximity of a cysteine and a lysine residue in the active site of 6-phosphogluconate dehydrogenase from Candida utilis.

The environment of a cysteine residue in the active site of 6-phosphogluconate dehydrogenase from Candida utilis was investigated by means of N-(3-pyrene) maleimide. The reaction between enzyme and inhibitor results in the modification of one cysteine residue per enzyme subunit, and in the complete inactivation of the enzyme. In a second step, the epsilon-amino group of a lysine residue causes an intramolecular aminolysis of the bound inhibitor. These results indicate that a lysine and a cysteine residues are close in the three-dimensional structure of the active site of 6-phosphogluconate dehydrogenase.

Non-enzymic protein phosphorylation. Phosphorylation of 6-phosphogluconate dehydrogenase by acyl phosphates.

Incubation of 6-phosphogluconate dehydrogenase from Candida utilis with either acetyl phosphate, 1,3-diphosphoglycerate or carbamoyl phosphate results in the phosphorylation of the protein. The binding of one phosphate residue per enzyme subunit does not affect significantly the kinetic properties, but makes the enzyme less reactive toward thiol reagents, trypsin and pyridoxal 5'-phosphate. We suggest indicate that: (1) 6-phosphogluconate dehydrogenase from C. utilis is phosphorylated non-enzymically by physiological acyl phosphates and (2) the phosphorylation of the enzyme modifies the rate of protein inactivation.

Substrate-induced intramolecular proton transfer in 6-phosphogluconate dehydrogenase from Candida utilis.

Formation of binary complex between 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP+ 2-oxidoreductase (decarboxylating), EC 1.1.1.44) from Candida utilis and 6-phosphogluconate was investigated by means of ultraviolet difference spectroscopy. The formation of the enzyme-substrate complex induces in the difference spectrum a positive peak the wavelength and extinction coefficient of which agree well with a tyrosine ionization. Titrimetric studies indicate that the formation of the binary complex is not coupled to a proton release from the protein. These data support an intramolecular proton transfer from a tyrosine to other functional group. This proton transfer could be correlated to the conformational change induced by substrate in 6-phosphogluconate dehydrogenase.

Effect of the substrate on the binding of coenzyme and coenzyme analogues to 6-phosphogluconate dehydrogenase from Candida utilis.

6-Phosphogluconate dehydrogenase from Candida utilis is a dimeric enzyme with apparently identical subunits. Two coenzyme analogues, periodate-oxidized NADP+ and 3-amino pyridine adenine dinucleotide phosphate, bind to the protein with Kdiss of 42 microM and 6.2 microM, respectively. NADPH binds to the enzyme with a Kdiss of 0.42 microM. Both coenzyme and coenzyme analogues show "all-of-the sites reactivity." In the presence of the substrate 6-phosphogluconate, the Kdiss of the coenzyme analogues is lowered to 3.5 microM and 2.4 microM, respectively, whereas the Kdiss of NADPH is increased to 2.7 microM. Coenzyme analogues, but not NADPH, show an half of-the sites reactivity in the presence of 6-phosphogluconate. The enzymatic activity is inhibited by high NADP+ concentrations in presence of low concentrations of substrate, whereas at high substrate concentrations no inhibition by NADP+ is showed. These data suggest that: 1) the enzyme exhibits an half-of-the-sites reactivity during the catalytic cycle; 2) the recycle of the enzyme occurs mainly through an abortive ternary complex enzyme-substrate-NADPH.

[[[Purification and properties of 6-phosphogluconate dehydrogenase from human erythrocytes]. / Purificazione e proprietà della 6-fosfogluconato deidrogenasi da eritrociti umani.

6-phosphogluconate dehydrogenase was purified from human erythrocytes by chromatography on 2'5' ADP Sepharose and ammonium sulfate precipitation, to a specific activity of 28 IU/mg of protein. The enzyme binds two NADP molecules per molecule of dimer with a Kd of 6.95 microM, or only one molecule of NADPH per molecule of dimer with a Kd of 0.38 microM. The substrate has no effect on the binding of NADPH, whereas increases the binding of a coenzyme analogue, the NADP oxidized with periodate. These findings indicate a possible regulatory role for 6-phosphogluconate dehydrogenase.