Decreased 13-S-hydroxyoctadecadienoic acid levels and 15-lipoxygenase-1 expression in human colon cancers.

13-S-Hydroxyoctadecadienoic acid (13-S-HODE), the product of 15-lipoxygenase (15-LOX) metabolism of linoleic acid, enhances cellular mitogenic responses to certain growth factors. Other observations have questioned whether 13-S-HODE has tumorigenic effects. Our study evaluated the hypothesis that 15-LOX-1 is overexpressed in colon cancers resulting in an increase in intracellular 13-S-HODE. 15-LOX-1 and 13-S-HODE were quantified using western blots, ELISA and immunohistochemistry in 18 human colon cancers with paired normal colonic mucosa. Additionally, 15-LOX-1 expression was measured by western blots in three transformed colonic cell lines and in a human umbilical vein endothelial cell line. Next, we evaluated 13-S-HODE effects on cellular proliferation, cell cycle distribution and apoptosis in a transformed colonic cell line (RKO). Cell cycle distributions were measured by flow cytometry and apoptosis was assessed by phase contrast microscopy, electron microscopy, flow cytometry and DNA fragmentation assay. 15-LOX-1 immunohistochemistry staining scores were reduced in tumor tissues (P

Mutations in the ER-Golgi intermediate compartment protein ERGIC-53 cause combined deficiency of coagulation factors V and VIII.

Combined deficiency of factors V and VIII is an autosomal recessive bleeding disorder resulting from alterations in an unknown gene on chromosome 18q, distinct from the factor V and factor VIII genes. ERGIC-53, a component of the ER-Golgi intermediate compartment, was mapped to a YAC and BAC contig containing the critical region for the combined factors V and VIII deficiency gene. DNA sequence analysis identified two different mutations, accounting for all affected individuals in nine families studied. Immunofluorescence and Western analysis of immortalized lymphocytes from patients homozygous for either of the two mutations demonstrate complete lack of expression of the mutated gene in these cells. These findings suggest that ERGIC-53 may function as a molecular chaperone for the transport from ER to Golgi of a specific subset of secreted proteins, including coagulation factors V and VIII.

Influence of human breast development on the growth properties of primary cultures.

In experimental animal models the susceptibility of the mammary gland to neoplastic transformation is related to its degree of development and proliferative activity; this observation led us to determine whether the human breast epithelium also exhibits development-related differences, and whether these differences could be detected in an in vitro system. Normal breast tissue obtained from reduction mammoplasties of 9 patients ranging in age from 18 to 56 years were characterized in both whole mount preparations and organoids obtained after collagenase-hyaluronidase digestion by their degree of development based upon the types of lobules present. Lobules were classified into type 1 (Lob 1), composed of approximately 11 alveolar buds, the less developed; lobules type 2 (Lob 2), of moderate development, composed of approximately 47 ductules each, and lobules type 3 (Lob 3), composed of 80 ductules each, represented the highest level of development. Epithelial organoids obtained after digestion were plated in DMEM:F12 medium supplemented with hydrocortisone, cholera toxin, insulin and 5% horse serum with a calcium concentration of 1.05 mM Ca++. Following attachment, the medium was replaced by medium containing 0.040 mM Ca++. The percentage of attachment of organoids to the flask was greater in cells from Lob 1 (89-99%) and Lob 1+2 (79-100%) than in cells from Lob 3, which had a 53-67% attachment. The total yield of cells after 7 weeks in culture was also greater in cells derived from Lob 1 and Lob 1+2 than in cells from Lob 3. The total yield of cells obtained from primary cultures was not related to the number of organoids plated, but to the degree of development of the gland. The DNA-labeling index (DNA-LI) in intact breast tissue correlated with that in primary cultures; it was greater in Lob 1 and Lob 1+2 than in Lob 3. By flow cytometry, the highest percentage of cells in S-phase was seen in cells with the highest DNA-LI. We concluded that the growth characteristics of mammary epithelial cells in vitro in a low Ca++ medium is modulated by the degree of development and differentiation of the gland.