p75 neurotrophin receptor inhibits invasion and metastasis of gastric cancer

The p75 neurotrophin receptor (p75NTR) is a focus for study at present. However, its function in gastric cancer was not elucidated. Here, we investigated its relation with metastasis of gastric cancer. By immunohistochemistry, we found that the positive rate of p75NTR expression in metastatic gastric cancer was 15.09% (16 of 106), which was lower compared with nonmetastatic gastric cancer (64.15%; 68 of 106). The average staining score in nonmetastatic gastric cancer was significantly higher than in metastatic gastric cancer (1.21 +/- 0.35 versus 0.23 +/- 0.18; P<0.01). p75NTR protein level was also lowly expressed in the highly liver-metastatic gastric cancer cell line XGC9811-L compared with other gastric cancer cell lines by Western blotting. It could also significantly inhibit the in vitro adhesive, invasive, and migratory and in vivo metastatic abilities of gastric cancer cell lines SGC7901 and MKN45 by reducing urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP)-9 proteins and by increasing tissue inhibitor of matrix metalloproteinase (TIMP)-1 protein. Further studies showed that p75NTR could suppress the nuclear factor-kappaB (NF-kappaB) signal. SN50, a specific inhibitor of NF-kappaB, which could inhibit in vitro invasive and migratory abilities of gastric cancer cells, reduced expression of uPA and MMP9 proteins and increased expression of TIMP1 protein. Taken together, p75NTR had the function of inhibiting the invasive and metastatic abilities of gastric cancer cells, which was mediated, at least partially, by down-regulation of uPA and MMP9 proteins and up-regulation of TIMP1 protein via the NF-kappaB signal transduction pathway. Our studies suggested that p75NTR may be used as a new potential therapeutic target in metastatic gastric cancer.     

Stimulation of Tubulin Synthesis by Lactate in Isolated Spermatogenic Cells

The effect of lactate on synthesis of new proteins in isolated spermatids and spermatocytes of rats was examined. Lactate stimulated[³⁵S]methionine ([³⁵S]met) incorporation into both spermatids and spermatocytes. The rate of protein synthesis was positively correlated with the intracellular level of ATP. The [³⁵S]met-labeled proteins in the two types of cells were compared by one and two dimensional polyacrylamide gel electrophoresis (1D and 2D-PAGE) and autoradiography. The syntheses of several stagespecific and non-specific proteins were observed. When spermatids and spermatocytes were cultured in medium without lactate, two major proteins of molecular weight (Mr) 43 kD and 55 kD were detected in the water-soluble fraction (105,000 g supernatant), and one major protein of Mr 24 kD was observed in the membrane-rich fraction. Addition of lactate to the incubation medium dramatically increased the synthesis of six proteins (Mr 14 kD, 16 kD, 43 kD, 55 kD, 84 kD and 135 kD) in the water-soluble fractions of spermatids and spermatocytes, but did not stimulate the synthesis of the Mr 24 kD protein in the membrane-rich fraction. In addition, after 1D and 2D-PAGE and electrophoretic transfer to nitrocellulose, two proteins of Mr 43 kD and 55 kD were identified as actin and tubulin, respectively, on the basis of their reactivities with specific antisera. Tubulin was also produced by in vitro translation using a spermatid lysate. These results suggest that lactate may play an important role in changing the cell structure and shape during spermatogenesis by regulating the syntheses of actin and tubulin.     

Identification of novel, stage-specific polypeptides associated with the differentiation of mammary epithelial stem cells to alveolar-like cells in culture

A linear pathway of morphologically intermediate cells has been identified between the cuboidal epithelial stem cells and the doming alveolar-like cultures of the cell line Rat Mammary (Rama) 25 in the order: cuboidal----grey----dark----dark droplet cell----doming cultures. The overall process can be accelerated by dimethyl sulfoxide (DMSO) or retinoic acid (RA) in the presence of mammotrophic hormones. From 400-450 [35S]methionine-labeled polypeptides that are routinely separated by two-dimensional gel electrophoresis approximately only 3% change during this process. As the Rama 25 cultures become confluent, three polypeptides of molecular weights (MW) 35 kD (pl = 7.7), 45 kD (pl = 7.5) and 33 kD (pl = 7.7) increase dramatically in radioactive abundance. These increases correspond to increases in numbers of grey cells for the 35 kD polypeptide, to increases in numbers of dark cells together with increases in peanut lectin-binding-ability for the 45 kD polypeptide, and to increases in the numbers of dark cells and in the numbers of droplet cells for the 33 kD polypeptide. After treatment with DMSO, RA or in spontaneously doming cultures, a second set of four polypeptides of MW 26 kD (pl = 5.9), 27 kD (pl = 6.2), 30 kD (pl = 7.2), and the same 33 kD polypeptide as above increase with the increase in numbers of droplet cells, domes, and increase in casein secretion. A variant of Rama 25, Rama 259, which fails to produce droplet cells, domes, or to secrete casein with DMSO and hormones also shows the same changes in the first set but not in the second set of polypeptides. The elongated, myoepithelial-like cell line derived from Rama 25, Rama 29, which cannot undergo any of the above intercellular conversions, fails to show changes in any of these polypeptides. Major changes in radioactive polypeptides have been confirmed for nonradioactive polypeptides and for polypeptides labeled for 4 hr with [35S]methionine. The synthesis of these novel polypeptides thus marks specific morphological stages of the differentiation of mammary epithelial stem to alveolar-like cells in culture, and as such may mark similar differentiation stages in vivo.     

Cell surface sialylation and tumor metastasis. Metastatic potential of B16 melanoma variants correlates with their relative numbers of specific penultimate oligosaccharide structures

Numerous investigations suggest that cell surface glycoconjugates, and in particular sialic acids, are directly involved in determining the metastatic phenotype. To further evaluate this hypothesis, we have used a variety of techniques to probe the cell surfaces of several metastatic variants of the murine B16 melanoma that were selected for experimental lung-colonizing ability (Fidler, I. (1973) Nature 242, 148-149) or for their ability to spontaneously metastasize from the site of a subcutaneous injection (Stackpole, C. W., Alterman, A. L., and Fornabaio, D. M. (1985) Invasion & Metastasis 5, 125-142). Using a highly sensitive high performance liquid chromatography sialic acid assay in conjunction with Vibrio cholerae sialidase, we find that none of these metastatic variants differ significantly in their overall levels of cell surface sialic acid. Using highly purified, linkage-specific sialyltransferases, in conjunction with specific glycosidases, to probe the cell surface saccharide topography of specific penultimate oligosaccharides, we also find no significant differences between the efficient lung-colonizing variant, B16-F10 and the poorly-colonizing B16-F1 or B16-Flr variants. In contrast, the spontaneously metastatic variants examined contain substantially different levels of specific penultimate sialylation sites. The tumorigenic but nonmetastatic B16-LM3/G3.26 variant contains 4-fold more penultimate Gal beta 1-3GalNAc sialylation sites than the tumorigenic and highly metastatic B16-LM3/G3.12 variant when CMP[3H]NeuAc and the alpha 2-3Gal beta 1-3GalNAc sialyltransferase are used to probe the melanoma cell surfaces. Several prominent glycoconjugates of apparent Mr 43,000, 40,000, and 30,000 are especially evident upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the nonmetastatic cells. The nonmetastatic variant also contains 2-fold more Gal beta 1-4GlcNAc sialylation sites than the metastatic variant when the alpha 2-6Gal beta 1-4GlcNAc sialyltransferase is used as a cell surface probe. In this case, glycoconjugates of apparent Mr 74,000, 45,000, and 43,000 are more prominently observed on the cell surfaces of the nonmetastatic variant. These data indicate that the differences in lung-colonizing abilities of B16 melanoma metastatic variants do not correlate with the numbers or sialylation states of specific penultimate oligosaccharide structures on their surfaces. However, the relative levels of specific penultimate saccharide structures do correlate with the ability of the cells to undergo spontaneous metastasis from a subcutaneous tumor.     

ICAM-2 expression mediates a membrane-actin link, confers a nonmetastatic phenotype and reflects favorable tumor stage or histology in neuroblastoma

The actin cytoskeleton is a primary determinant of tumor cell motility and metastatic potential. Motility and metastasis are thought to be regulated, in large part, by the interaction of membrane proteins with cytoplasmic linker proteins and of these linker proteins, in turn, with actin. However, complete membrane-to-actin linkages have been difficult to identify. We used co-immunoprecipitation and competitive peptide assays to show that intercellular adhesion molecule-2 (ICAM-2)/alpha-actinin/actin may comprise such a linkage in neuroblastoma cells. ICAM-2 expression limited the motility of these cells and redistributed actin fibers in vitro, and suppressed development of disseminated tumors in an in vivo model of metastatic neuroblastoma. Consistent with these observations, immunohistochemical analysis demonstrated ICAM-2 expression in primary neuroblastoma tumors exhibiting features that are associated with limited metastatic disease and more favorable clinical outcome. In neuroblastoma cell lines, ICAM-2 expression did not affect AKT activation, tumorigenic potential or chemosensitivity, as has been reported for some types of transfected cells. The observed ICAM-2-mediated suppression of metastatic phenotype is a novel function for this protein, and the interaction of ICAM-2/alpha-actinin/actin represents the first complete membrane-linker protein-actin linkage to impact tumor cell motility in vitro and metastatic potential in an in vivo model. Current work focuses on identifying specific protein domains critical to the regulation of neuroblastoma cell motility and metastasis and on determining if these domains represent exploitable therapeutic targets.     

Proportion of antigenic variants induced by in vitro UV irradiation differs in clones derived from a single tumor

The purpose of this study was to examine the capacity of different clones derived from the same tumor to generate highly antigenic cells after in vitro exposure to UV radiation. Cells from the metastatic murine melanoma K1735 and clones of K1735 differing in metastatic potential were exposed to UV radiation in vitro, cloned, and tested for antigenic properties in vivo. Approximately half of the clones isolated after UV irradiation of parental K1735 melanoma cells were highly antigenic (five of nine). Similar treatment of cells of a nonmetastatic clone of K1735 generated clones that were all antigenic (nine of nine). In contrast, only one of nine clones derived from UV-irradiated cells of a highly metastatic clone of K1735 were antigenic. Clones derived from unirradiated cultures were not antigenic variants. The increased antigenicity of cells derived from UV-irradiated cultures did not correlate with an increase in expression of cell surface class I major histocompatibility complex antigens. These results demonstrate that the frequency of antigenic variant production after UV irradiation is an intrinsic property of the particular cell line used, and that even cloned cell lines derived from a single tumor differ in their ability to generate antigenic variants after UV irradiation. In addition, they indicate that the increased antigenicity is not necessarily due to a UV-induced increase in expression of cell surface class I histocompatibility antigens.     

Characterization of the major proteins of bovine seminal fluid by two-dimensional polyacrylamide gel electrophoresis

Recently, we demonstrated that the major proteins from bovine seminal plasma BSP-A1, -A2, -A3 and -30-kDa (collectively called BSP proteins) specifically interact with choline phospholipids. These proteins coat the surface of the spermatozoa after ejaculation and are believed to play an important role in membrane modifications occurring during capacitation. In this study we determined the isoelectric point (pl) and analysed the molecular heterogeneity of BSP proteins. Total protein from bovine seminal plasma (CBSP) and purified BSP proteins were iodinated using chloramine T. Samples were reduced, denatured, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and visualized by autoradiography. Analysis of CBSP proteins demonstrated the presence of polypeptides migrating in the pH range of 3.5–7.8 and at molecular weights (Mr) between 6 and 100 kDa. Many isoforms of each BSP protein were found when purified iodinated proteins were analysed by 2D-PAGE. BSP-A1 was found at a Mr of 16.5 kDa and in the range of pl of 4.7–5.0; BSP-A2 at 16 kDa and at a pl of 4.9–5.2; BSP-A3 at 15 kDa and at a pl of 4.8–5.2, and BSP-30-kDa at 28 kDa and at a pl of 3.9–4.6. Similar results were obtained with immunolocalization of BSP proteins after Western blot using specific antibodies. The treatment of purified iodinated BSP proteins with neuraminidase increased the pl of BSP-30-kDa to 4.8–5.0 and decreased its Mr to 25 kDa, but no change was observed for BSP-A1, -A2 and -A3. The treatment of BSP proteins with sulfatase or acid phosphatase modified neither their Mr nor their pl. Furthermore, when CBSP proteins were separated in 2D-PAGE and the gels stained for glycoproteins with dansyl hydrazine, BSP proteins were among the major glycoproteins found in the bovine seminal plasma. In conclusion, BSP proteins are acidic and have several isoforms. Furthermore, the heterogeneity of BSP-30-kDa is mainly due to its sialic acid content. © 1994 Wiley-Liss, Inc.     

Comparison of protein synthesis patterns in mouse cumulus cells and mural granulosa cells: effects of follicle-stimulating hormone and insulin on granulosa cell differentiation in vitro.

Successful development of mammalian oocytes requires correct interactions between developing oocytes and associated granulosa cells. Development of oocyte-granulosa cell complexes from preantral follicles in vitro does not produce oocytes competent to develop to blastocysts at the same frequency as for oocytes that develop in vivo. Addition of either FSH or insulin to cultures of oocyte-granulosa cell complexes does not improve the frequency of blastocyst development, and the combination of both insulin and FSH is deleterious. Here, high-resolution 2-dimensional PAGE (2D-PAGE) and computerized gel image analysis were used to compare patterns of protein synthesis in cumulus cells and mural granulosa cells of small antral follicles, and then to assess effects of FSH and insulin on the differentiation of oocyte-associated granulosa cells (OAGCs) in vitro. Culture of OAGCs without FSH or insulin resulted in failure to synthesize many proteins at rates characteristic of cumulus cells. Either hormone used alone caused many cumulus cell proteins that were decreased in control cultures to be synthesized at nearly normal cumulus cell rates, and also caused the synthesis of other proteins to be increased or decreased. The two hormones added together produced the greatest change in protein synthetic pattern, including overexpression or underexpression of many proteins not affected by either hormone alone. Addition of these hormones to culture media thus appeared insufficient to elicit a normal cumulus cell phenotype in OAGCs and could lead to complex changes in protein synthesis that may be deleterious to oocyte development. The high-resolution 2D-PAGE approach described here should be a valuable tool in studies on oocyte and granulosa cell development in vitro, since phenotype can be evaluated globally through the display of over 1000 newly synthesized proteins rather than relying upon the expression of just a few genes.     

Genomic analysis of a spontaneous model of breast cancer metastasis to bone reveals a role for the extracellular matrix

A clinically relevant model of spontaneous breast cancer metastasis to multiple sites, including bone, was characterized and used to identify genes involved in metastatic progression. The metastatic potential of several genetically related tumor lines was assayed using a novel real-time quantitative RT-PCR assay of tumor burden. Based on this assay, the tumor lines were categorized as nonmetastatic (67NR), weakly metastatic to lymph node (168FARN) or lung (66cl4), or highly metastatic to lymph node, lung, and bone (4T1.2 and 4T1.13). In vitro assays that mimic stages of metastasis showed that highly metastatic tumors lines were more adhesive, invasive, and migratory than the less metastatic lines. To identify metastasis-related genes in this model, each metastatic tumor was array profiled against the nonmetastatic 67NR using 15,000 mouse cDNA arrays. A significant proportion of genes relating to the extracellular matrix had elevated expression in highly metastatic tumors. The role of one of these genes, POEM, was further investigated in the model. In situ hybridization showed that POEM expression was specific to the tumor epithelium of highly metastatic tumors. Decreased POEM expression in 4T1.2 tumors significantly inhibited spontaneous metastasis to the lung, bone, and kidney. Taken together, our data support a role for the extracellular matrix in metastatic progression and describe, for the first time, a role for POEM in this process.     

Subtractive immunization yields monoclonal antibodies that specifically inhibit metastasis

Subtractive immunization allowed the isolation and characterization of monoclonal antibodies that specifically inhibit metastasis but not proliferation of highly metastatic human tumor cells. The tolerizing agent cyclophosphamide was used to suppress the immune system in mice to dominant immunodeterminants present on a non-metastatic variant (M-) of the human epidermoid carcinoma cell line (HEp3). Mice were then inoculated with a highly metastatic variant (M+) of HEp3 to enhance an immune response to antigenic determinants present on metastatic cells. Hybridomas were generated and screened by ELISA for differential reactivity to M+ HEp3 over M- HEp3 cells. This experimental approach, termed subtractive immunization (S.I.), was compared to a control immunization protocol, which eliminated the cyclophosphamide treatment. The S.I. protocol resulted in an eight-fold increase in the proportion of mAbs that react with molecules enriched on the surface of the M+ HEp3 cells. Two of the mAbs derived from the S.I. protocol, designated DM12-4 and 1A5, were purified and examined for their effect in a metastasis model system in which chick embryos are transplanted with primary HEp3 tumors. Purified mAbs DM12-4 and 1A5, inoculated i.v. into the embryos, inhibited spontaneous metastasis of HEp3 cells by 86 and 90%, respectively. The mAbs are specifically anti-metastatic in that they have no effect on the growth of HEp3 cells in vitro nor did they inhibit primary tumor growth in vivo. The mAbs recognize M+ HEp3 cell surface molecules of 55 kD and 29 kD, respectively. These data demonstrate that the S.I. protocol can be used for the development of unique mAbs that are reactive with antigenic determinants whose expression is elevated on metastatic human tumor cells and which function mechanistically in the metastatic cascade.     

Association of the metastatic phenotype with CCN family members among breast and oral cancer cells

The CCN family of proteins consists of six members with conserved structural features. These proteins play several roles in the physiology and pathology of cells. Among the pathological roles of the CCN family, one of the most important and controversial ones is their role in the expansion and metastasis of cancer. Up to now a number of reports have described the possible role of each CCN family member independently. In this study, we comprehensively analyzed the roles of all six CCN family members in cell growth, migration and invasion of breast cancer cells in vitro and in vivo. As a result, we found the CCN2/CCN3 ratio to be a parameter that is associated with the metastatic phenotype of breast cancer cells that are highly metastatic to the bone. The same analysis with cell lines from oral squamous carcinomas that are not metastatic to the bone further supported our notion. These results suggest the functional significance of the interplay between CCN family members in regulating the phenotype of cancer cells.     

Autocrine and paracrine regulation of tissue inhibitor of metalloproteinases, transin, and urokinase gene expression in metastatic and nonmetastatic mammary carcinoma cells.

Acquisition of metastatic competence by tumor cells is frequently accompanied by increased expression of extracellular proteases capable of degrading basement membrane and extracellular matrix. However, very little is known about how the genes encoding these enzymes and their inhibitor proteins are regulated in metastatic versus nonmetastatic cells. In this report, we have compared autocrine and paracrine regulation of tissue inhibitor of metalloproteinases (TIMP), transin, and urokinase plasminogen activator (uPA) genes in genetically related nonmetastatic SP1 and metastatic A3a cell lines. Compared to SP1 cells, metastatic A3a cells showed 15-20-fold higher transin, 3-5-fold less TIMP mRNA, and comparable levels of uPA mRNA. A qualitatively similar shift in expression of these genes was rapidly (i.e., 4-8 h) induced in nonmetastatic SP1 cells following the addition of conditioned medium from A3a cells. The gene-regulating activity present in A3a conditioned medium was heat-labile, suggesting that it was protein in nature. The responsiveness of SP1 cells to the factor(s) secreted by A3a conditioned medium was inhibited by cycloheximide. Basic fibroblast growth factor mimicked the effect of the A3a conditioned medium as an inducer of transin expression in the tumor cells. Although medium conditioned by the tumor cells did not affect uPA expression, addition of epidermal growth factor to the tumor cells transiently induced expression of uPA with a biphasic response that differed in SP1 and A3a cells. Initial induction of uPA at 2-4 h was similar for both cell lines, but after 24 h of exposure to epidermal growth factor, SP1 cells showed a net reduction in uPA, whereas metastatic cells returned to the unstimulated levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of the alpha7beta1 laminin receptor suppresses melanoma growth and metastatic potential.

The alpha7beta1 integrin is a laminin-binding receptor that was originally identified in melanoma. Here, we show that, in clonally derived mouse K1735 melanoma variant cell lines with high (M-2) and low (C-23) metastatic potential, elevated expression of alpha7 correlates with reduced cell motility, metastasis, and tumor growth. Both cell lines showed similar beta1 integrin-dependent adhesion to laminin-1 and the E8 laminin fragment. However, the highly metastatic M-2 cells rapidly migrated on laminin, whereas the nonmetastatic C-23 cells were minimally motile. Laminin-binding integrin profiles showed that the M-2 cells expressed moderate amounts of alpha1 and abundant alpha6 but low or undetectable levels of alpha2 and alpha7. By contrast, C-23 cells expressed low or undetectable levels of alpha1, alpha2, and alpha6 but had up-regulated levels of alpha7. Consistent with the protein data, Northern blot analysis showed that levels of alpha7 mRNA were highest in the poorly metastatic variant cells, whereas alpha6 message was not detected; in contrast, alpha6 mRNA was elevated in the highly metastatic cells, whereas alpha7 message was not detected. Forced expression of alpha7 in the M-2 cells suppressed cell motility, tumor growth, and metastasis. Collectively, these results indicate that, during melanoma progression, acquisition of a highly tumorigenic and metastatic melanoma phenotype is associated with loss of the alpha7beta1 laminin receptor.     

Spontaneous fusion in vivo between normal host and tumor cells: possible contribution to tumor progression and metastasis studied with a lectin-resistant mutant tumor.

Previous studies demonstrated that growth in DBA/2 mice of MDW4, a wheat germ agglutinin-resistant (WGAr) mutant of the highly metastatic MDAY-D2 DBA/2 mouse tumor, led to the emergence of WGA-sensitive (WGAs) revertants having higher ploidy levels at the site of inoculation as well as at distant visceral metastases. The results implied that MDW4 was nonmetastatic but progressed to become metastatic in vivo only after a cellular change took place which was accompanied by extinction of the WGAr phenotype and acquisition of a higher number of chromosomes. Results presented here provide strong and direct evidence for the underlying mechanism being spontaneous cell fusion in vivo between the MDW4 (WGAr) tumor cells and normal host cells, at least some of which are of bone marrow origin. Thus, growth of the H-2d MDW4 tumor cells in (C3H X DBA/2)F1 (H-2k X H-2d) or (C57BL/6 X DBA/2)F1 (H-2b X H-2d) mice led to the appearance of WGAs revertants bearing the H-2k or H-2b major histocompatibility complex antigens associated with the C3H or C57BL/6 parental strains, respectively. Similarly, WGAs revertants of MDW4 were found to express H-2k antigens after growth in CBA/HT6T6 (H-2k) leads to DBA/2 bone marrow radiation chimeras. Attempts to mimic the in vivo hybridization process were successful in that in vitro somatic cell fusion between an ouabain-resistant (OuaR), 6-thioguanine-resistant (Thgr) derivative of the MDW4 mutant and either normal bone marrow or spleen cells resulted in loss of the WGAr phenotype in the hybrids (thus showing its recessive character) and increased malignant properties in vivo. An analysis of spontaneous frequencies of re-expression of various drug resistance genetic markers in several hybrid metastatic cells was also consistent with chromosome segregation of the sensitive alleles. The results show that tumor progression and the emergence of metastatic cell variants could arise as a consequence of tumor X host cell fusion followed by chromosome segregation. We also discuss the possibility that this type of event may normally be a very rare one during the growth of tumors, the frequency of which can be artificially amplified by the use of certain classes of lectin-resistant mutants carrying particular cell surface alterations.     

Exposure of human primary colon carcinoma cells to anti-Fas interactions influences the emergence of pre-existing Fas-resistant metastatic subpopulations

Fas, an important death receptor-mediated signaling pathway, has been shown to be down-regulated during human colon tumorigenesis; however, how alterations in Fas expression influence the metastatic process remains unresolved. In mouse models, loss of Fas function was found to be both necessary and sufficient for tumor progression. In this study, we investigated the link between functional Fas status and malignant phenotype using a matched pair of naturally occurring primary (Fas-sensitive) and metastatic (Fas-resistant) human colon carcinoma cell lines in both in vitro and in vivo (xenograft) settings. Metastatic sublines were produced in vitro from the primary tumor cell line by functional elimination of Fas-responsive cells. Conversely, sublines derived from the primary tumor in vivo at distal metastatic sites were Fas-resistant. In contrast, simply disrupting the Fas pathway by molecular-based strategies in the Fas-sensitive primary tumor failed to achieve the same metastatic outcome. Interestingly, both in vitro- and in vivo-produced sublines resembled the naturally occurring metastatic population, based on functional and morphologic studies and genome-scale gene expression profiling. Overall, using this human colon carcinoma model, we: 1) showed that loss of Fas function was linked to, but alone was insufficient for, acquisition of a detectable metastatic phenotype; 2) demonstrated that metastatic subpopulations pre-existed within the heterogeneous primary tumor, and that anti-Fas interactions served as a selective pressure for their outgrowth; and 3) identified a large set of differentially expressed genes distinguishing the primary from metastatic malignant phenotypes. Thus, Fas-based interactions may represent a novel mechanism for the biologic or immunologic selection of certain types of Fas-resistant neoplastic clones with enhanced metastatic ability.     

A requirement for dimerization of HP1Hsalpha in suppression of breast cancer invasion

The development and progression of cancer is controlled by gene expression, often regulated through chromatin packaging. Heterochromatin protein 1(Hsalpha) (HP1(Hsalpha)), one of three human HP1 family members, participates in heterochromatin formation and gene regulation. HP1(Hsalpha) possesses an amino-terminal chromodomain, which binds methylated lysine 9 of histone H3 (meK9 H3), and a carboxyl-terminal chromoshadow domain (CSD) that is required for dimerization and interaction with partner proteins. HP1(Hsalpha) is down-regulated in invasive metastatic breast cancer cells compared with poorly invasive nonmetastatic breast cancer cells. Expression of EGFP-HP1(Hsalpha) in highly invasive MDA-MB-231 cells causes a reduction in in vitro invasion, without affecting cell growth. Conversely, knock-down of HP1(Hsalpha) levels in the poorly invasive breast cancer cell line MCF-7 increased invasion, without affecting cell growth. To determine whether functions of the CSD were required for the regulation of invasion, mutant forms of HP1(Hsalpha) were expressed in MDA-MB-231 cells. A W174A mutation that disrupts interactions between HP1(Hsalpha) and PXVXL-containing partner proteins reduced invasion similar to that of the wild type protein. In contrast, an I165E mutation that disrupts dimerization of HP1(Hsalpha) did not decrease invasion. No gross changes in localization and abundance of HP1(Hsbeta), HP1(Hsgamma), and meK9 H3 were observed upon expression of wild type and mutant forms of HP1(Hsalpha) in MDA-MB-231 cells. Taken together, these data demonstrate that modulation of HP1(Hsalpha) alters the invasive potential of breast cancer cells through mechanisms requiring HP1 dimerization, but not interactions with PXVXL-containing proteins.