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We examined bone marrow aspirates from 100 metastasis-free primary breast cancer patients. In 38/100 patients (38%), tumour cells were detected in the marrow using an immunocytochemical technique with a cocktail of two monoclonal antibodies: anti-EMA and anti-cytokeratin. Median follow-up was 34 months: 15/38 (39%) tumour cell-positive patients have since relapsed, but only 9/62 (15%) tumour cell-negative patients. The median interval between tumour cell detection and relapse was 11.4 months. No statistically significant correlation existed between tumour cell presence and 'established' prognostic factors. However, relapse-free survival was significantly shorter in tumour cell-positive patients. Multivariate analysis showed tumour cell presence as a strong, significant prognostic factor for relapse-free as well as overall survival. We conclude that screening for tumour cells in bone marrow of primary breast cancer patients identifies high-risk patients for early relapse. In particular, patients with node-negative tumours who have tumour cells in their bone marrow may require subsequent systemic therapy.

<h4>Background</h4>The molecular genetic changes associated with colorectal carcinoma are among the best understood of any common human cancer. The genetic changes during the late stages of colorectal carcinomas may be useful in clinical management for determining the metastatic potential of the carcinoma.<h4>Methods</h4>Tumor tissues were evaluated by restriction fragment length polymorphism (RFLP) analysis of chromosomes 5q, 17p, 18q, and 22q (n = 98), by reverse transcription-polymerase chain reaction (RT-PCR) analysis of messenger RNA expression of the DCC gene (deleted in colorectal carcinoma) (n = 27) and by immunohistochemical analysis of p53 protein expression (n = 44).<h4>Results</h4>Loss of heterozygosity (LOH) on chromosomes 17p, 18q, and 22q, but not on 5q, was much more frequently detected in advanced carcinomas than in intramucosal carcinomas (P < 0.01). 17p LOH was significantly correlated with vascular invasion (P < 0.001), whereas 18q LOH was correlated with lymphatic invasion and hepatic metastasis (P < 0.01), and 22q LOH was correlated with lymph node metastasis (P < 0.05). LOH on 5q did not show a significant correlation with any factors of tumor invasion or metastasis. DCC expression was not observed in any of five hepatic metastasis or in five of seven advanced carcinomas that were accompanied by hepatic metastasis (10 of 12). However, a similar lack of expression was observed in only 5 of 15 carcinomas without hepatic metastasis (P < 0.05). p53 Expression was found to vary in both primary and metastatic carcinomas by immunohistochemistry.<h4>Conclusions</h4>The clinical application of molecular genetics (i.e., RFLP analysis of chromosome 17p, 18q, and 22q and RT-PCR analysis of DCC expression into messenger RNA) can be used to determine the metastatic potential of colorectal carcinomas.

Two 2-year-old Salers cattle from different herds raised on pasture were evaluated for retarded growth and diarrhea. Increase of liver enzyme activities and prolonged sulfobromophothalein (BSP) half life (T1/2) indicated liver disease with impaired liver function. Histopathologic examination of liver biopsies revealed a micronodular cirrhosis with marked deposition of hemosiderin in hepatocytes, Kupffer cells, and arterioles. Transferrin saturation (TS) and liver iron content were markedly increased, consistent with a diagnosis of hemochromatosis. Both animals were euthanatized due to deterioration in their condition. Necropsy findings included hepatomegaly and hemosiderin accumulation in the liver, lymph nodes, pancreas, spleen, thyroid, kidney, brain and other glandular tissue. Continued surveillance of the second herd (serum iron, total iron binding capacity [TIBC], unsaturated iron binding capacity [UIBC], and TS), identified a heifer as a hemochromatosis suspect in a subsequent generation. Liver biopsies from that animal revealed the same histopathologic changes as the previous 2 animals, and similar increases in liver iron content (8,700 ppm, normal range 45 to 300 ppm). The 3 affected cattle were all products of line breeding programs and shared a common ancestor. The absence of dietary iron loading in conjunction with the histopathologic and metabolic findings were consistent with a diagnosis of primary hemochromatosis. The reported disease is similar to idiopathic hemochromatosis in human beings in which there is a hereditary defect in iron metabolism.

To determine if the MCC, DCC or p53 gene is associated with susceptibility to hereditary non-polyposis colorectal cancer (HNPCC), these genes in normal cells from 12 HNPCC patients were analysed by polymerase chain reaction-single strand conformation polymorphism analysis. No changes which may alter the amino acid sequences of these genes were detected, suggesting that these genes are not associated with the susceptibility to HNPCC. Only one of nine HNPCC cancers showed mutations in the MCC and p53 genes on the same analysis. Loss of heterozygosity in chromosomes 5q, 17p, 18q and 22 was detected in four of the nine cancers, all of them being positive as to metastasis to lymph nodes. Abnormalities of the (CA)n repeat were found in six cancers, including all four without metastasis. These data indicate that tumor suppressor genes in chromosomes 5q, 17p, 18q and 22 are associated with the late stage of colorectal tumorigenesis in HNPCC, whereas the (CA)n repeat abnormalities are an early event of tumorigenesis and more essential to HNPCC.

In recent years, identifying the hepatic cell type responsible for collagen synthesis in experimental models of postnecrotic or inflammatory fibrosis has been the subject of active investigation. In primary iron overload states, however, hepatic fibrosis and cirrhosis occur without accompanying necroinflammatory phenomena. In this study, we combined morphological, immunological, cell isolation and purification and molecular biological techniques to identify the hepatic cell responsible for enhanced collagen type I gene expression during chronic enteral iron overload in the rat. Ultrastructural analysis of liver tissue sections from iron-loaded rats specifically revealed an altered appearance of fat-storing cells, which showed few if any fat droplets left and increased rough endoplasmic reticulum. In situ hybridization analysis with specific complementary RNA probes identified enhanced signal for collagen type I into nonparenchymal cells in zones 1 and 2, without signal over the background onto iron-laden hepatocytes. Immunocytochemistry with desmin antibodies combined with in situ hybridization on the same tissue sections identified the cells expressing high level of collagen type I transcripts as fat-storing cells. Northern-blot analysis on RNA extracted from various purified cell isolates, confirmed the presence of collagen type I mRNA signal only into the fat-storing cells isolate. Our study shows that in an experimental model of metabolic fibrosis in which the hepatotoxin selectively accumulates into parenchymal cells, fat-storing cells are the main source of enhanced collagen type I gene expression.

The goal of the study was to identify any normal genes that may become inactivated in malignant cells, with associated modifications or loss of gene products. Consequently, attempts were made to identify such products by generating monoclonal antibodies using an immune tolerisation-immunisation procedure. Using such a technique, a plasma membrane-associated glycoprotein with an apparent molecular weight of 92 kDa was identified. The glycoprotein was termed luminal epithelial antigen (LEA.92). The pattern of expression of LEA.92 was demonstrated by an indirect immunostaining technique. Using an in vitro model system representing various stages of breast oncogenesis, LEA.92 was detected on normal or immortalised mammary epithelial cell (MEC) lines which were dependent on epidermal growth factor (EGF) and anchorage formation for growth and non-tumorigenic in nude mice. In contrast, LEA.92 was undetectable on oncogenically transformed or established lines of mammary carcinoma cell lines which were independent of EGF or anchorage formation for growth and were highly tumorigenic. The results appear to suggest a correlation between the down-regulation of LEA.92 and the development of tumorigenicity in malignant MEC lines. Furthermore, the patterns of expression of LEA.92 on breast cells in tissue mirrored those of breast epithelial cells in cell cultures. LEA.92 was detected on the surface of normal but not malignant epithelial cells, which included breast, cervix, colon, lung, pancreas and stomach. LEA.92 appeared to be distinct from receptor for epidermal growth factor, antigens associated with milk fat globule membrane and the family of epithelium-specific keratins.

Commercially available immunoradiometric assays were used for pS2 and total cathepsin D determination in the cytosol fraction obtained from 266 primary breast cancers. We show that pS2 and cathepsin D values were significantly associated (Spearman's rank correlation: P < 0.0001) in tumours from lymph node-positive patients (N+), while such association did not reach significance in tumours taken from patients with negative lymph nodes (N-). Moreover, cathepsin D concentrations in pS2-rich tumours (pS2 above the median value, 5 ng mg-1 protein) were significantly higher (Mann-Whitney-Wilcoxon's rank-sum test: P = 0.00001) than those obtained in the samples expressing less than 5 ng of pS2 per mg of protein. pS2 was also correlated to both the oestrogen receptor (ER) (Spearman's rank correlation: P < 0.0001) and the progesterone receptor (PR) (Spearman's rank correlation: P = 0.022). No significant differences in the expression of pS2 and cathepsin D taken from N+ and N- patients were found. Furthermore, no significant differences in pS2 and cathepsin D expression were obtained by stratifying tumours on the basis of their size (T). pS2 and cathepsin D values obtained in ER-positive/PR-positive tumours did not significantly differ from the values obtained in ER-positive/PR-negative and in ER-negative/PR-positive tumours. We conclude that pS2 could have a role in cathepsin D expression, and that it can be used in the assessment of a functioning oestrogen response machinery in those tumours that express only ER.

In this study, we investigated the effects of luteinizing hormone-releasing hormone (LH-RH) agonist [D-Trp6]LH-RH, LH-RH antagonist [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH-RH (SB-75), and estradiol on the growth of human epithelial ovarian cancer cell line OV-1063. Cells were cultured under estrogen-deprived conditions. Estradiol inhibited cell proliferation, as measured by cell number at 10(-9)-10(-7) M and [3H]thymidine incorporation into DNA at 10(-13)-10(-8) M. Both LH-RH analogs inhibited cell growth dose dependently in the range 10(-8)-10(-5) M, but SB-75 induced a greater growth inhibition than [D-Trp6]LH-RH. In OV-1063 cells, 125I-labeled [D-Trp6]LH-RH was bound to one class of specific, saturable binding sites with high affinity (Kd = 1.4 +/- 0.3 nM) and low capacity (4000 binding sites per cell). 125I-labeled [D-Trp6]LH-RH could be displaced by unlabeled [D-Trp6]LH-RH and SB-75, suggesting that both analogs are bound to the same receptor on OV-1063 cells. Ligand binding was dependent on time and temperature. Receptor internalization assay showed that the ligand-receptor complex was internalized at 37 degrees C, which indicates the presence of biologically active LH-RH receptors on OV-1063 cells. These results suggest that estradiol and LH-RH analogs can suppress the growth of OV-1063 human epithelial ovarian cancer cells by a direct action and that the inhibitory effect of LH-RH analogs is mediated through the high-affinity LH-RH receptors.

We describe the isolation, growth-suppressing activity, and chromosomal localization of the human homologue of the murine growth-arrest-specific gene gas1. Overexpression of h-gas1 is able to block cell proliferation in the A549 lung carcinoma and the T24 bladder carcinoma cell lines. No effect was observed when h-gas1 was introduced into the osteosarcoma cell line SAOS-2 and into the adenovirus-type-5 transformed cell line 293. This finding is related to our previous evidence that simian virus 40-transformed NIH 3T3 cells are also refractory to murine gas1 overexpression, suggesting that the retinoblastoma and/or p53 gene products have an active role in mediating the growth-suppressing effect of gas1. We also show that h-gas1 is on chromosome 9q21.3-22.1, in a region considered to be a fragile site. Altogether, the results raise the possibility that h-gas1 may be a target for genetic alterations leading to its inactivation in tumor cells.

The Deleted in Colorectal Cancer (DCC) gene is a candidate tumor suppressor gene that is predicted to encode a transmembrane polypeptide with strong similarity to the neural cell adhesion molecule (N-CAM) family. Previous studies have suggested that several different N-CAMs, when expressed in non-neuronal cell types can stimulate neurite outgrowth from PC12 rat pheochromocytoma cells. Based on the predicted structural similarity of DCC to N-CAMs, we sought to determine whether NIH3T3 cells expressing DCC could stimulate neurite outgrowth in PC12 cells. We found that NIH3T3 cell lines expressing DCC could stimulate PC12 cells to extend neurites. Supernatants from DCC-transfected NIH3T3 cells did not induce neurite outgrowth above background levels, suggesting that cell-cell interaction was required. NIH3T3 cells expressing a truncated form of DCC, lacking the majority of the cytoplasmic domain sequences, also failed to induce neurite outgrowth above the levels seen with control NIH3T3 cells, suggesting that the cytoplasmic domain of DCC was necessary for its neurite-promoting function. In contrast to NGF-mediated neurite outgrowth, the DCC-mediated response was inhibited by treatment with pertussis toxin or the combination of N- and L-type calcium channel blockers, and was unaffected by the transcriptional inhibitor cordycepin. The data suggest that the DCC protein can function in a fashion analogous to other N-CAMs to alter PC12 cell phenotype through intracellular pathways distinct from those involved in NGF signaling.

We describe the spontaneous progression of a colon adenoma cell line to tumorigenicity and growth factor independence. This system allows direct comparison of biologic stages of malignant progression with alterations of colon cancer suppressor genes and oncogenes. VACO-235, a human colon adenoma cell line, is at early passages nontumorigenic in the nude mouse, unable to grow in soft agar, growth stimulated by serum and EGF, and growth inhibited by TGF-beta. VACO-235 daughter passages 93 and higher have in culture spontaneously progressed to being weakly tumorigenic, but retain all other growth characteristics of VACO-235 early passages. A mouse xenograft from late passage VACO-235 was reestablished in culture as the granddaughter cell line, VACO-411. VACO-411 is highly tumorigenic, clones in soft agar, and is unresponsive to serum, EGF, and TGF-beta. Early passage VACO-235 bears a mutant K-ras allele, bears only mutant APC alleles, expresses no DCC transcripts, and expresses only wild type p53 transcripts. VACO-411 retains the identical genotype, still expressing only wild type p53. Colonic cells after ras mutation, APC mutation, and DCC inactivation remain nontumorigenic and growth factor dependent. Malignant progression involves at least two additional steps, and in VACO-411 can proceed by a novel pathway not requiring p53 inactivation.

Src homology 3 (SH3) domains are found in numerous cytoplasmic proteins involved in intracellular signal transduction. We used 2-D 1H NMR to determine the structure of the SH3 domain of the guanosine triphosphatase-activating protein (GAP), an essential component of the Ras signaling pathway. The structure of the GAP SH3 domain (275-350) was found to be a compact beta-barrel made of six antiparallel beta-strands arranged in two roughly perpendicular beta-sheets with the acidic residues located at the surface of the protein. The Trp317, Trp319, Thr321 and Leu323 residues belonging to the sequence (317-326), which was shown to be essential for Ras signaling, formed two nearby lipophilic bulges followed by a hydrophilic domain (Arg324-Asp326). These structural data could be used to characterize the still unidentified downstream components of GAP, which are involved in Ras signaling, and to rationally design inhibitors of this pathway.

<h4>Aims</h4>(i) To assess the validity of an immunocytochemical technique for detecting pS2 protein in paraffin wax embedded tissue; (ii) to provide further data on the relation between pS2 protein and oestrogen receptor (ER) and progesterone receptor (PgR).<h4>Methods</h4>Breast cancer excision biopsy specimens were obtained from 35 previously untreated patients. An immunoradiometric assay was compared with an immunohistochemical method for measuring pS2 protein. ER and PgR were measured in cytosol fractions by enzyme immunoassay and the relation between the presence of these receptors and pS2 protein was assessed before and after subdivision of the women into groups of over or under 50 years of age.<h4>Results</h4>A good correlation was seen between the immunoradiometric and immunohistochemical methods for pS2 protein measurement (r = 0.84; p = 0.0001). Log-transformed data showed a significant correlation between increasing values of ER and pS2 protein (r = 0.45; p = 0.006) and to a lesser extent between pS2 protein and PgR (r = 0.38; p = 0.03). Correlations were also shown between pS2+ and PgR+ status (p = 0.01), and between ER and PgR positivity (p = 0.05; Fisher's exact test). pS2+ protein status was only associated with ER+ status in patients aged 50 years or less.<h4>Conclusions</h4>The two methods for pS2 analysis are virtually interchangeable. This provides strong support for using immunohistochemistry for pS2 in paraffin wax embedded tissue. The association with ER positivity and pS2+ protein status only in the premenopausal patients may be due to the higher levels of oestrogenic stimuli in that group.

To assess the contribution of thrombus formation in the pathogenesis of unstable angina, we employed the recently developed assays of small fragments which reflect the degree of activation of various components of the haemostatic system. Such haemostatic measurements were undertaken in patients with unstable angina (n = 47) from the time of their admission to the coronary care unit (CCU) at 8-h intervals in the first 24 h and then daily for a total of 5 days. The results obtained were compared with healthy control values. Patients exhibited lower ATIII, prolongation of the APTT and TT, but not PT or the reptilase time, which is a consequence of heparinization. There was significant elevation of fibrinogen, factor VIII:C, von Willebrand factor:antigen and von Willebrand factor:ristocetin cofactor throughout the study period. There was also evidence of thrombin generation as indicated by the elevated levels of fibrinopeptide A (FPA) and thrombin-antithrombin complexes. The platelet release proteins, beta-thromboglobulin (BTG) and platelet factor 4 (PF4), were markedly elevated in the first 2 days and dropped gradually thereafter. The fibrinolytic inhibitor, plasminogen activator inhibitor (PAI), levels were elevated throughout. Proteins C and S, plasminogen and alpha 2-antiplasmin remained unchanged. It was concluded that in patients with unstable angina, there is significant activation of the clotting system and inhibition of fibrinolysis which confirms the existence of a tendency towards thrombus formation in patients with unstable angina.

The influence of 17 beta-estradiol (E2) and prolactin was studied on N-methyl-N-nitrosourea (MNU)-induced mammary carcinomas (MCAs) in rats. MNU was intravenously injected once into seven-week-old female. female F344 rats at a dose of 50 mg/kg body weight. Groups of rats also received either 2.5 mg of E2 or a continuous supply of prolactin and/or growth hormone via transplanted MtT/F84 (mammo-somatotropic pituitary tumor). Rats were observed for up to 36 weeks after MNU administration. Although simultaneous administration of MNU and E2 did not much affect the occurrence of MCAs as compared to administration of MNU alone, rats treated with 2.5 mg of E2 for two weeks before MNU administration had significantly reduced occurrence of MCAs compared to those given MNU alone. In contrast, rats with MNU plus MtT/F84 showed high incidence and shortened latency of MCAs and they also had a high incidence of clitorial gland hyperplasias. Average pituitary weights and serum prolactin levels in E2-treated rats were greatly increased compared to those of MNU-alone rats. Average serum E2 levels were about 100 ng/ml in E2-treated rats and 0.05 ng/ml in rats without E2 treatment. Serum prolactin levels were greatly increased in rats with MtT/F84. The results indicated that pretreatment with E2 before MNU administration was inhibitory while increased prolactin caused by grafting MtT/F84 after MNU injection was promotive for the occurrence of MCAs in female F344 rats.

In this study, we measured in vitro proliferative responses of peripheral blood mononuclear cells from both leprosy patients across the clinical spectrum and also healthy contacts from a leprosy-endemic population to delipidified cell components of Mycobacterium leprae (DCC) and Dharmendra lepromin. Dharmendra lepromin was poor in inducing in vitro T cell proliferation in all the study groups, even though it elicited marked in vivo skin test reaction in tuberculoid leprosy patients and healthy contacts. In contrast, Dharmendra preparation of BCG induced marked T-cell response in tuberculoid as well as bacterial index negative lepromatous patients. DCC induced a significantly higher lymphoproliferative response than Dharmendra lepromin in all study groups. A significant positive correlation was observed between the lymphoproliferative responses to DCC and BCG. The present study, based on a large number of leprosy patients and healthy contacts, clearly demonstrates that DCC, depleted of glycolipids and lipopolysaccharides, is a good antigenic preparation for evaluating T-cell reactivity to M. leprae.