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Women newly admitted to hospital for suspect breast lump (99) were blind studied 5+/-3 days before diagnosis. Depression was evaluated by using DSM-III-R diagnostic criteria and MMPI psychometric test; estrogen receptor (ER) by DCC. High prevalence of depressive mood disorders and particularly of dysthymia were observed in patients with ER(-) tumors (p=0.03), that scored low in MMPI 9-Ma scale (p<0.001) and high in TA index (p=0.01) as expression of depressed mood. On the contrary ER(+) patients showed higher mean values in almost all the MMPI scales and indexes (AV p<0.01; AS p<0.03). In conclusion depressive mood have different prevalence in untreated breast cancer patients depending on ER status.

In many cancers, inactivating mutations in both alleles of the transforming growth factor beta (TGF-beta) type 11 receptor (TbetaRII) gene occur and correlate with loss of sensitivity to TGF-beta. Here we describe a novel mechanism for loss of sensitivity to growth inhibition by TGF-beta in tumor development. Mac-1 cells, isolated from the blood of a patient with an indolent form of cutaneous T-cell lymphoma, express wild-type TbetaRII and are sensitive to TGF-beta. Mac-2A cells, clonally related to Mac-1 and isolated from a skin nodule of the same patient at a later, clinically aggressive stage of lymphoma, are resistant to TGF-beta. They express both the wild-type TbetaRII and a receptor with a single point mutation (Asp-404-Gly [D404G]) in the kinase domain (D404G-->TbetaRII); no TbetaRI or TbetaRII is found on the plasma membrane, suggesting that D404G-TbetaRII dominantly inhibits the function of the wild-type receptor by inhibiting its appearance on the plasma membrane. Indeed, inducible expression, under control of a tetracycline-regulated promoter, of D404G-TbetaRII in TGF-beta- sensitive Mac-1 cells as well as in Hep3B hepatoma cells results in resistance to TGF-beta and disappearance of cell surface TbetaRI and TbetaRII. Overexpression of wild-type TbetaRII in Mac-2A cells restores cell surface TbetaRI and TbetaRH and sensitivity to TGF-beta. The ability of the D404G-TbetaRH to dominantly inhibit function of wild-type TGF-beta receptors represents a new mechanism for loss of sensitivity to the growth-inhibitory functions of TGF-beta in tumor development.

Chromosome deletions are the most common genetic events observed in cancer. These deletions are generally thought to reflect the existence of a tumour suppressor gene within the lost region. However, when the lost region does not precisely coincide with a hereditary cancer locus, identification of the putative tumour suppressor gene (target of the deletion) can be problematic. For example, previous studies have demonstrated that chromosome 18q is lost in over 60% of colorectal as well as in other cancers, but the lost region could not be precisely determined. Here we present a rigorous strategy for mapping and evaluating allelic deletions in sporadic tumours, and apply it to the evaluation of chromosome 18 in colorectal cancers. Using this approach, we define a minimally lost region (MLR) on chromosome 18q21, which contains at least two candidate tumour suppressor genes, DPC4 and DCC. The analysis further suggested genetic heterogeneity, with DPC4 the deletion target in up to a third of the cases and DCC or a neighbouring gene the target in the remaining tumours.

<h4>Purpose</h4>To report African Americans with primary iron overload diagnosed during life and to study iron stores in African Americans undergoing autopsy.<h4>Patients and methods</h4>We summarized information for 4 African-American patients diagnosed during life with iron overload not explainable by alcohol, blood transfusions, or ineffective erythropoiesis. We reviewed liver specimens and hospital records of 326 unselected adult African Americans who were autopsied, assessing Prussian blue-stained sections for hepatocellular iron and measuring iron quantitatively in specimens that stained positively. We calculated the hepatic iron index (the hepatic iron concentration in mumol/g dry weight divided by the age in years). In autopsy subjects we corrected the index to account for iron administered by blood transfusion (the adjusted hepatic iron index). The hepatic iron index is useful for distinguishing primary iron overload from the moderate siderosis that may accompany alcoholic liver disease. The normal index is < or = 1.0. An index > or = 1.7 cannot be explained by alcohol effects and an index > or = 1.9 indicates the magnitude of iron-loading found in Caucasian homozygous HLA-linked hemochromatosis.<h4>Results</h4>The 4 living patients, all males and 27 to 50 years of age, had elevated body iron burdens and one or more of the following: hepatomegaly, cirrhosis, cardiomyopathy, diabetes mellitus, and impotence. Hepatic iron indices were 2.3, 11.5, and 20.2 in the 3 whose liver iron concentrations were measured. Among the autopsy subjects, 4 (1.2%), 2 men and 2 women aged 50 to 63 years, had adjusted hepatic iron indices > or = 1.9 (range 1.9 to 5.6).<h4>Conclusions</h4>Primary iron overload occurs in African Americans. Further studies are needed to define prevalence, pathophysiology and clinical consequences. Clinicians should look for this condition.

It has been reported that several genes may be good indicators for determining biological behavior, including the prognosis, of colorectal cancers. We have summarized these reported genes, such as tumor suppressor gene, oncogenes, metastasis suppressor gene, adhesion molecules, growth factors, proteinases, and others, including microsatellite instability. Some of the genes such as p53, DCC, c-met, or matrix metalloproteinase are considered to be reliable for determining biological aggressiveness. We introduced several interesting genes which we are focusing using cDNA subtraction library analysis. We hope that these genes are well combined for best analysis of the biological behavior of colorectal cancers and use for practical clinical analysis. In addition, we hope that novel important genes indicative for prognosis will be found.

Inactivation of the p53 tumour-suppressor gene is common in a wide variety of human neoplasms. In the majority of cases, single point mutations in the protein-encoding sequence of p53 lead to positive immunohistochemistry (IHC) for the p53 protein, and are accompanied by loss of the wild-type allele. Recently, the WAF1/Cip1 gene was identified as one of the genes induced by wild-type p53, and increased expression of p21WAF1/Cip1 has been found to reflect the status of the p53 tumour-suppressor pathway. We investigated the inactivation of p53 in a relatively small, but well-characterised, group of 46 colorectal carcinomas that were previously studied for allelic alterations, ras oncogene mutations and DNA aneuploidy. Alterations in p53 were identified by IHC, loss of 17p and DNA sequence analysis of exons 5-8, whereas p21WAF1/Cip1 protein expression was determined by IHC. p53 mutations were identified in 19 of the 46 tumours (41%), whereas positive IHC for p53 was found in 21 of the 46 tumours (46%). Positive IHC for p21WAF1/Cip1 was detected in 16 of 42 cases (38%). We found no relationship between p21WAF1/Cip1 staining and p53 protein expression or p53 mutational status. Inactivating mutations in the p53 gene correlated with LOH at 17p but not with LOH at 5q or 18q, Dukes' stage, tumour grade or DNA ploidy. There was a higher survival rate independent of Dukes' stage in the group with no alterations in p53 compared with those with evidence of dysfunction of p53, but the difference was not statistically significant. We conclude that inactivation of p53 and altered expression of p21WAF1/Cip1 are common in colorectal carcinoma but do not correlate with each other or with the clinical or pathological parameters investigated.

During acute inflammation, P-selectin is transiently mobilized from Weibel-Palade bodies to the surface of histamine-activated endothelial cells, where it mediates rolling adhesion of neutrophils under hydrodynamic flow. During chronic or allergic inflammation, sustained expression of P-selectin on the endothelial cell surface has been observed. We found that the cytokines interleukin 4 (IL-4) or oncostatin M (OSM) induced a five- to ninefold increase in P-selectin messenger RNA (mRNA) in human umbilical vein endothelial cells (HUVEC) that persisted as long as 72 h. IL-4 elevated P-selectin mRNA by increasing its transcription rate rather than by prolonging its already long half-life. Stimulation of P-selectin transcription by IL-4 or OSM required new protein synthesis and tyrosine phosphorylation of cellular proteins. Tumor necrosis factor alpha, IL-1 beta, lipopolysaccharide, or IL-3 did not increase P-selectin mRNA in HUVEC, and did not augment the IL-4-induced increase in P-selectin transcripts. IL-4 or OSM increased P-selectin protein on the cell surface as well as in Weibel-Palade bodies. Under flow conditions, neutrophils rolled on P-selectin expressed by IL-4-treated HUVEC, and even more neutrophils rolled on P-selectin after IL-4-treated HUVEC were stimulated with histamine. These data demonstrate that IL-4 or OSM stimulates endothelial cells to synthesize more P-selectin over prolonged periods. The increased expression of P-selectin may facilitate the emigration of leukocytes into sites of chronic or allergic inflammation.

We evaluate the relation between genotype and phenotype in 47 Italian male patients with homozygous genetic hemochromatosis (GH). Phenotype evaluation was based on the ratio of amount of iron removed (IR) by phlebotomy and age (IR/age). Patients were divided in two classes of phenotype expression: class I included 26 patients with less severe iron overload (IR/age <0.33) and class II included 21 patients with a more marked one (IR/age >0.33). Genetic variability was assessed by haplotype analysis combining alleles at HLA-B, D6S265, HLA-A, and D6S105 loci. A common ancestral haplotype carrying D6S265-1, HLA-A3, and D6S105-8 alleles was present in 13 of 52 (25%) chromosomes in class I and in 24 of 42 (57%) chromosomes in class II (P = .0027). Homozygotes and heterozygotes for the ancestral haplotype had higher iron indices than patients carrying two haplotypes other than the ancestral one. Seven of the eight patients homozygous for the ancestral haplotype were in class II, heterozygotes were equally distributed between the two classes, whereas 14 of 18 carriers of other haplotype combinations were in class I. Our results suggests that the gene defect linked to the ancestral haplotype is the result of a single, severe mutation. The high variability of phenotype expression in heterozygotes for the ancestral haplotype could be accounted for the contribution of the mutation carried by the second haplotype. Combination of different mutations could be responsible for the variable degrees of iron overload found in patients with GH.

Cell adhesion to the extracellular matrix is largely mediated by adhesion molecules of the integrin family and is often diminished upon oncogenic transformation. However, we show here that the chronic myelogenous leukemia oncogene Bcr/Abl has positive effects on VLA-4 and VLA-5 integrin function. The presence of Bcr/Abl in the GM-CSF- or IL-3-dependent hematopoietic cell lines MO7e, 32D, and BaF/3 enhanced cell binding to both soluble and immobilized fibronectin. The effect was due to enhanced function of the VLA-5 integrin fibronectin receptor and not to increased surface expression. In parallel, Bcr/Abl stimulated cell adhesion to the VLA-4 integrin ligand VCAM-1. Stimulation of VLA-5 function directly correlated with induction of Bcr/Abl tyrosine kinase activity in a temperature-sensitive kinase mutant. Thus, Bcr/Abl stimulates integrin-dependent cell adhesion, by a mechanism involving increased ligand binding, with the tyrosine kinase activity of Bcr/Abl likely playing a key role. Consistent with these results, hematopoietic precursor cells from chronic myelogenous leukemia patients also showed increased adhesion to fibronectin.

This study concerns DNA ploidy, numerical changes of chromosomes 7, 8, 10, 17 and 18, and allelic losses at chromosomes 17p13.3 (flanking the p53 gene) and 18q21 (location of the DCC gene) in 31 freshly resected colorectal tumours. Cytological smears were used to determine DNA ploidy by image analysis, and chromosome numbers by fluorescence in situ hybridization (FISH) using chromosome-specific pericentromeric alpha-satellite DNA probes. Allelic losses were assessed by Southern blotting and by the polymerase chain reaction loss of heterozygosity method. Approximately 50% of the tumours were aneuploid. There was heterogeneity with respect to chromosome numbers, but gains and losses of chromosomes, or both, were detected in all carcinomas examined, including 10 that were nonaneuploid by image analysis. Trisomy 7 was found in 74% of the tumours, and monosomy of chromosome 18 in 32%. Allelic loss at chromosome 17p13.3 was evident in 13 of 26 informative cases, and only one case exhibited monosomy 17. In comparison monosomy 18 was found in 10 cases; 7 of them corresponded to approximately half of the cases with allelic loss within the DCC gene, and the other three were noninformative. These findings indicate that the loss of one chromosome 18 is an important mechanism producing allelic deletion of the DCC gene in colorectal carcinomas. Our data also suggest that monosomy 18 is a useful indicator for studying colorectal cancer progression on a cell by cell basis.

Karyotyping and polymorphisms within the DCC (deleted in colon cancer) locus (18q21) were used to analyze loss of chromosome 18 in squamous cell carcinomas (SCC). Tumors from 26 patients (including 7 for whom matched tumor and normal DNA samples were available) were examined for heterozygosity within DCC. Of the seven normal-tumor combinations, four were informative. Two of these had loss of heterozygosity (LOH) at DCC. For 19 SCC tumor cultures normal tissue was not available. These were scored only as homozygous or heterozygous. The majority were homozygous. Only 3/19 (15%) were heterozygous. In contrast, in a panel of normal blood samples the majority, 11/16 (69%), were heterozygous. Allelic zygosity was concordant with the chromosome 18 content in the 16 tumors that were also karyotyped. Tumors from 40 patients, 37 that were karyotyped and three that were informative at the DCC locus, were assessed for loss of chromosome 18 and patient survival. Loss of part or all of chromosome 18 occurred in tumors from 25. Twenty-seven of the 40 patients have died and 13 are alive. There was strong association between loss of 18 and overall survival. Of those who are alive only 5/13 (38%) had loss of 18, whereas among those who have died 20/27 (74%) had loss of 18. By chi 2 analysis the association of loss of 18 and death from cancer was significant (p > 0.01). The high frequency of chromosome 18 loss in SCC suggests that this region contains one or more tumor suppressor genes important in the clinical behavior of SCC. DCC is one candidate, but other regions of loss not including the DCC locus indicate that chromosome 18 probably contains more than one tumor suppressor locus. Prospective studies of chromosome 18 loss as a single prognostic indicator are strongly indicated in this tumor type since loss in early stage tumors might indicate a need for more aggressive therapy than would be given on the basis of staging alone.

We examined the effect of the synthetic pentasaccharide representing the minimal binding site of heparin to antithrombin on the antithrombin-mediated inactivation of factor VIIa bound to tissue factor. This effect was compared to the effect of unfractionated heparin. Using purified recombinant human coagulation factors and either a clotting or an amidolytic assay for the determination of the residual activity of factor VIIa, we showed that the pentasaccharide was an efficient antithrombin-dependent inhibitor of the coagulant activity of tissue factor-factor VIIa complex. In our experimental conditions, assuming a mean MW of 14,000 for heparin, the molar pseudo-first order rate constants for ATIII-mediated FVIIa inhibition by ATIII-binding heparin and by the synthetic pentasaccharide were found to be similar with respective values of 104,000 +/- 10,500 min-1 and 112,000 +/- 12,000 min-1 (mean +/- s.e.m., n = 3).

Development in higher organisms involves complex genetic regulation at the molecular level. The emerging picture of development control includes several families of master regulatory genes which can affect the expression of down-stream target genes in developmental cascade pathways. One new family of such development regulators is the SOX gene family. The SOX genes are named for a shared motif called the SRY box a region homologous to the DNA-binding domain of SRY, the mammalian sex determining gene. Like SRY, SOX genes play important roles in chordate development. At least a dozen human SOX genes have been identified and partially characterized (Tables 1 and 2). Mutations in SOX9 have recently been linked to campomelic dysplasia and autosomal sex reversal, and other SOX genes may also be associated with human disease.

The preparation of a new class of isothiocyanatopeptide bifunctional chelating agents, Boc-glycyl-L-(p-NCS)phenylalanylglycylglycine ethyl ester 1, N-(S-acetylmercaptoacetyl)-L-(p-NCS)phenylalanylglycylglycine ethyl ester 2, and N-(S-acetylmercaptoacetyl)glycyl-L-(p-NCS)phenylalanylglycine ethyl ester 3, starting from Boc-L-(p-nitro)phenylalanine 5 is described. The key intermediates, nitropeptides 7, 8 and 12, were prepared by standard DCC coupling and a deprotection procedure. The nitropeptides 7 and 12 on condensation with N-succinimidyl-S-acetylthioacetate provided a good yield of the corresponding S-(acetylmercaptoacetyl)nitropeptides 10 and 13, respectively. Catalytic hydrogenolysis of compounds 8, 10 and 13, followed by thiophosgenyation gave 28-95% yield of target products 1, 2 and 3, respectively. Compounds 1-3 were conjugated to the monoclonal antibody D612 reactive with human colon carcinoma using 12/1 to 60/1 ligand to antibody molar ratios, and the conjugates were labeled with 99mTc. The in vitro cell binding results of these immunoconjugates demonstrate that they retained their antibody binding specificity.

Abnormalities of coagulation and fibrinolysis were studied in a group of 28 children and young adults with homozygous sickle cell disease (SCD), either in the steady state (n = 12) or during painful crisis (n = 16). Coagulation was explored by standard clotting tests and by measurement of prothrombin complex factors, factor VIII (VIII:C) and antithrombin III (ATIII), protein C (PC) and protein S (PS) activities, while fibrinolytic potential was evaluated using D-dimer, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) assays. In SCD patients, thrombin time (TT) was constantly shortened, both in the steady state (ratio to control 0.83 +/- 0.08, p < 0.0001) and in crisis (0.76 +/- 0.06, p < 0.0001). Mean levels of prothrombin complex were similar in asymptomatic patients to those in controls, but were significantly decreased during sickle cell crisis (p < 0.05 for factor V and p < 0.0001 for factors II, VII and X). Factor VIII:C was significantly increased, both in the steady state (207 +/- 35%, p < 0.0001) and during crisis (208 +/- 34%, p < 0.0001). PS activity was reduced int he steady state (81 +/- 12%, p < 0.01) and further diminished in crisis (68.5 +/- 27.5%, p < 0.001), while D-dimers were significantly elevated during sickle cell crisis (1028 +/- 675 ng/ml, p < 0.001). In all SCD patients, baseline levels of t-PA antigen were comparable to those in controls, whereas concentrations of PAI-1 antigen were significantly increased, either in the steady state (89.7 +/- 26.3 ng/ml, p < 0.0001) or in crisis (75.0 +/- 24.8 ng/ml, p < 0.0001). These results provide evidence for the presence of circulating activated clotting factors in SCD and for an imbalance of the profibrinolytic and antifibrinolytic systems most likely due to increased PAI-1 levels.

DCC gene located at chromosome 18q21.3 is an important tumor-supressor gene associated with tumorigenesis and progression, especially in colorectal carcinomas. Adopting Southern blot technique and RFLP analysis, 33 primary Chinese colorectal cancer patients were examined for loss of heterozygosity (LOH) of DCC gene. Among 16 informative cases, 6 (38%) showed allelic deletion. 18q LOH was correlated with lymph node and hepatic metastases (P < 0.05). The results indicate that application of molecular genetics is of value in ascertaining the metastatic potential of colorectal carcinomas.

To evaluate the role of APC, MCC and DCC genes in the development and progression of colorectal cancers, loss of heterozygosity (LOH) at these genetic loci was investigated in 41 surgically resected specimens of colorectal cancer by using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. LOH at APC or/and MCC was detected in 38.9% (14/38) of the informative cases; at the APC in 28.0% (7/25) and at MCC locus in 36.4% (8/22). LOH at DCC genetic locus was detected in 55.3% (21/38) of the informative cases. LOH rates DCC gene in the cancer specimens with lymphnode metastasis (80.0%) and in Dukes' stage III and IV (71.4%) were significantly higher than that in specimens without lymphnode metastasis (39.1%) and in Dukes' stage I and II (35.3%) (P < 0.05). Our results suggest that LOH at APC/MCC and DCC is the common genetic alterations in colorectal cancer and DCC genetic LOH assay may be useful in predicting the prognosis of patients with colorectal cancer.

Both deleted in colorectal carcinoma (DCC) and mutated in colorectal carcinomas (MCC) genes are newly found tumour suppresser genes in colorectal carcinomas. In this study mutations of DCC gene and loss of heterozygosity (LOH) of MCC gene were detected with Southern blot and PCR methods respectively. The study consisted of 30 cases of colorectal adenoma (10 with mild, 10 with moderate and 10 with severe dysplasia) and 33 cases of colorectal carcinomas (13 highly, 10 moderately and 10 poorly differentiated, meanwhile 9 in Dukes A, 10 in B and 14 in C stage). Their paired normal colorectal mucosa served as control. The results were as follows: mutation of DCC was not found in any specimen with normal mucosa or with adenoma of mild and moderate dysplaaia, while it was found in one if those with severe dysplasia. Mutation of DCC was, however, found in 7 of the 33 specimens with carcinoma and there was a correlation between the positive rate and the differentiation or staging of the carcinoma. LOH of MCC was not found in normal mucosa, but it was found in 2/10, 3/10 and 4/10 of the adenoma specimens with mild, moderate and severe dysplasia respectively. 18/33 of the carcinoma specimens was found to have LOH of MCC and there was also a correlation between the positive rate and the differentiation or staging of the carcinoma. These results suggest that the frequency of multiple gene changes increase with poor differentiation and increased staging of colorectal carcinoma.

DCC, a candidate tumor suppressor gene from chromosome 18q21, is most highly expressed in the developing nervous system. In vitro studies suggest a role for DCC in neuronal differentiation, and 18q allelic loss occurs in a subset of neuroblastomas. To address the hypothesis that loss of DCC function may contribute to tumorigenesis in cells of neural origin, we utilized a combination of RNase protection, immunoblotting, and immunohistochemical approaches to characterize DCC expression in 62 primary neuroblastomas and 16 neuroblastoma cell lines. The DCC protein was undetectable in 38% of the primary tumors and 56% of the cell lines. Of note, primary tumors lacking DCC expression were more likely to have been obtained from patients with disseminated or stage D disease (P = 0.01). In addition, loss of DCC expression was observed in three of six primary tumors from stage DS patients. No consistent relationship between the loss of DCC expression and N-myc amplification was observed in our studies. Our findings suggest that loss of DCC expression may contribute to the dissemination of neuroblastoma cells, perhaps through alterations in growth and differentiation pathways distinct from those regulated by N-myc.