The goal of our study was to develop a panel of tumor cell lines along with paired non‐malignant cell lines or strains collected from breast cancers, predominantly primary tumors. From a total of 189 breast tumor samples consisting of 177 primary tumors and 12 metastatic tissues, we established 21 human breast tumor cell lines that included 18 cell lines derived from primary tumors and 3 derived from metastatic lesions. Cell lines included those from patients with germline BRCA1 and FHIT gene mutations and others with possible genetic predisposition. For 19 tumor cell lines, we also established one or more corresponding non‐malignant cell strains or B lymphoblastoid (BL) lines, which included 16 BL lines and 7 breast epithelial (2) or stromal (5) cell strains. The present report describes clinical, pathological and molecular information regarding the normal and tumor tissue sources along with relevant personal information and familial medical history. Analysis of the breast tumor cell lines indicated that most of the cell lines had the following features: they were derived from large tumors with or without axillary node metastases; were aneuploid and exhibited a moderate to poorly differentiated phenotype; were estrogen receptor (ER)‐ and progesterone receptor (PR)‐negative; and overexpressed p53 and HER2/neu proteins. Of 13 patients with primary breast cancers receiving curative intent mastectomies, 7 were dead after a mean period of 10 months. Our panel of paired tumor and non‐malignant cell lines should provide important new reagents for breast cancer research. Int. J. Cancer 78:766–774, 1998. © 1998 Wiley‐Liss, Inc.

During fertilization in Chlamydomonas, gametes of opposite mating types interact with each other through sex-specific adhesion molecules on their flagellar surfaces. Flagellar adhesion brings the cell bodies of the gametes into close contact and initiates a signal transduction pathway in preparation for cell–cell fusion. We have identified a cDNA, gsp1, whose transcript levels are upregulated during flagellar adhesion. The GSP1 polypeptide is a novel, gamete-specific homeodomain protein, the first to be identified in an alga. Its homeodomain shows significant identity with several higher plant homeodomain proteins. Although encoded by a single copy gene present in cells of both mating types, immunoblot analysis showed that GSP1 was expressed in mating type (mt)+ gametes, but was not detectable in mt− gametes or in vegetative cells of either mating type. Moreover, GSP1 appeared late during gametogenesis, suggesting that it may function during adhesion with mt− gametes or after zygote formation. GSP1 is expressed in imp11, mt− mutant gametes, which have a lesion in the mid gene involved in sex determination and exhibit many phenotypic characteristics of mt+ gametes. Thus, gsp1 is negatively regulated by mid and is the first molecule to be identified in Chlamydomonas that shows sex-limited expression.

Chlamydomonas gametes of opposite mating types interact through flagellar adhesion molecules called agglutinins leading to a signal transduction cascade that induces cell wall loss and activation of mating structures along with other cellular responses that ultimately result in zygote formation. To identify molecules involved in these complex cellular events, we have employed subtractive and differential hybridization with cDNA from mt+ gametes activated for fertilization and non-signaling, vegetative (non-gametic) cells. We identified 55 cDNA clones whose transcripts were regulated in activated gametes. Here we report the molecular cloning and characterization of the complementary DNA (cDNA) for one clone whose transcripts in activated gametes were several-fold higher than in normal gametes. Regulation of the transcript was not related simply to protein synthesis because it was not increased in cells synthesizing new cell wall proteins. The cDNA contained a single open reading frame (ORF) of 815 amino acids encoding a polypeptide of calculated relative mass of 87 kDa. Database search analysis and sequence alignment indicated that the deduced amino acid sequence exhibited 42% identity and 62% similarity to a class of prokaryotic methyl transferases (5-methyltetrahydrofolate-homocysteine methyl transferase; EC 2.1.1.14) known to be involved in the terminal step of de novo biosynthesis of methionine. This enzyme catalyzes transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine resulting in methionine formation. Affinity-purified polyclonal antibodies raised against a bacterially produced GST-fusion protein identified a 85 kDa soluble protein in Chlamydomonas gametes. Southern blot hybridization indicated that the enzyme is encoded by a single-copy gene. The evidence presented in this paper raises the possibility that, in addition to its participation in de novo biosynthesis and regeneration of methionine, Chlamydomonas methionine synthase may play a role in adhesion-induced events during fertilization.

In the unicellular eukaryote Chlamydomonas, disruption of cell-matrix interactions by treatment with a periplasmic matrix metalloproteinase, g-lysin, activates a signal transduction pathway that results in the rapid synthesis and secretion of matrix molecules, followed by their assembly into a new matrix. I have identified and partially characterized several cDNA clones for transcripts that are dramatically up-regulated following treatment of cells with g-lysin. Here I report the complete nucleotide sequence and preliminary characterization of a matrix-related molecule termed Mrp47. The cDNA clone for Mrp47 contained an insert of 2.5 kb, corresponding to a transcript of 3.0 kb that is encoded by a single-copy gene. Sequence analysis indicated that Mrp47 cDNA contains an open reading frame (ORF) that encodes a 46-kDa polypeptide. The putative polypeptide is unusually rich in the amino acids proline, alanine and serine, with prolines clustered together in a 30-amino acid N-terminal region and a 80-amino acid C-terminal region. Further analysis of the predicted amino acid sequence suggested that Mrp47 is likely to be a secreted glycoprotein. Southern hybridization analysis indicated that Mrp47 is encoded by a single-copy gene in the Chlamydomonas genome. Database searches suggested that Mrp47 shows homology to other proline-rich proteins including a surface glycoprotein in Volvox and verprolin from yeast.

Fertilization in Chlamydomonas is initiated by adhesive interactions between gametes of opposite mating types through flagellar glycoproteins called agglutinins. Interactions between these cell adhesion molecules signal for the activation of adenylyl cyclase through an interplay of protein kinases and ultimately result in formation of a diploid zygote. One of the early events during adhesion-induced signal transduction is the rapid inactivation of a flagellar protein kinase that phosphorylates a 48-kDa protein in the flagella. We report the biochemical and molecular characterization of the 48-kDa protein. Experiments using a bacterially expressed fusion protein show that the 48-kDa protein is capable of autophosphorylation on serine and tyrosine and phosphorylation of bovine .8-casein on serine, confirming that the 48-kDa protein itself has protein kinase activity. This protein kinase exhibits limited homology to members of the eukaryotic protein kinase superfamily and may be an important element in a signaling pathway in fertilization.Many biological processes are regulated by protein phosphorylation/dephosphorylation after receptor-ligand interactions at the cell surface. While there is evidence to suggest that cell-cell adhesion between gametes is translated into phosphorylation reactions inside the cell, with the exception of those involved in the yeast pheromone response and mating (1-3), no protein kinase with a biochemical role in fertilization has been identified. In the unicellular eukaryotic alga Chlamydomonas, adhesive interactions between receptor-like glycoproteins on gametic flagella of opposite mating types lead to the activation of a flagellar adenylyl cyclase (4-6) through a complex interplay of several protein kinase activities (6-8). One of these protein kinase activities, an ATP-dependent inhibitor of adenylyl cyclase, is proposed to maintain the adenylyl cyclase at basal levels of activity in nonadhering gametes (6)(7)(8). A second protein kinase activity is required for the adhesion-dependent activation of flagellar adenylyl cyclase (6). The activation of adenylyl cyclase in turn results in a rapid increase in intracellular cAMP levels, which trigger downstream cellular events including cell wall loss and mating structure formation in preparation for cell fusion (9).In experiments to identify adhesion-dependent changes in flagellar protein kinase activity, we found (Y.Z., Y.L., and W.J.S., unpublished data) that flagellar adhesion between gametes of opposite mating types during fertilization rapidly leads to inhibition of a flagellar protein kinase activity that phosphorylates a 48-kDa flagellar protein. In in vitro phosphorylation assays, the 48-kDa protein was phosphorylated in flagella isolated from nonadhering gametes, but its phosphorylation could not be detected in flagella isolated from gametes that had been adhering for 1 min. Available evidence indicates that the inactivation of this protein kinase may be an eventThe publication costs of this article were defrayed in part b...