Tracking prostate carcinoma micrometastasis to multiple organs using histochemical marker genes and novel cell systems.

Studies of human prostate carcinoma (PCA) have been hampered by only a few cell systems from already-metastatic human disease. We have developed a novel cell system by using tissue cultured CWR22R cells from a xenograft of a primary tumor from a human patient. These cells were transfected with the bacterial lacZ gene to maximize their detection during progression and metastasis in nude mice. LZ-CWR22R cells are extremely stable for lacZ expression over 25 passages and metastasize to lung, liver, and bone from the subcutis - major sites of metastasis of the human disease. A matrigel vehicle facilitated development of primary tumors and micrometastases in all organs. While some micrometastases developed into overt metastases, others remained as micrometastases for long periods of time, possibly providing a model of latency of metastatic disease. An experimental metastasis model (tail vein injection) also generated micrometastases in lung, liver, and bone with differing kinetics of formation and stability. Serial sections of many individual lung micrometastases within one hour of injection indicated considerable heterogeneity in cellular composition (from 1 to 19 cells/site) while liver sites at later times were comprised of only 1 or 2 cells (the size of bone sites were comparable to those of liver). By combining use of these histochemically-tagged PCA cell systems with high resolution molecular analyses (laser-capture microdissection), it will now be possible to analyze gene expression patterns characteristic of micrometastases developing in several different organs.

Tracking micrometastasis to multiple organs with lacZ-tagged CWR22R prostate carcinoma cells.

Metastasis to organs other than lung is rarely observed in animal model systems of human prostate carcinoma (PCA), with the exception of already metastatic isolates of human PCA cultured for long periods of time. To analyze more directly the evolution of metastatic variants from primary PCA tumor isolates, the lacZ histochemical marker gene was transfected into the CWR22Rv1 cell line isolated from the CWR22R xenograft (primary tumor). Three clones of varying lacZ-expression stability were analyzed for tumorigenicity and progression in athymic nude mice. Clones B and D were highly tumorigenic in the subcutis; however, lacZ expression was highly unstable. In contrast, clone H demonstrated highly stable lacZ expression for >25 passages in culture or in animals. Clone H, injected sc in a PBS vehicle, gave a 15-40% tumorigenic take. All primary tumor-bearing animals exhibited micrometastases in lung and other organs. Clone H injected in a Matrigel vehicle gave 100% tumorigenicity, with all animals displaying micrometastases in lung, liver, and/or bone (lower frequency in brain and kidney). Overall, the relative frequency of micrometastasis to multiple organs was lung>liver=bone>>brain>kidney. Overt metastases were never observed in the lung or bone but were occasionally found in liver. lacZ-transfected clone H CWR22Rv1 cells represent a much more accurate model of metastasis of PCA to the organs normally involved in progression of the human disease. Use of marker gene-tagged cells and other high-resolution molecular techniques will now permit analyses of the earliest events in PCA progression and micrometastasis.

Tagged tumor cells reveal regulatory steps during earliest stages of tumor progression and micrometastasis.

Histochemical marker genes were used to "tag" mouse fibrosarcoma or human neuroblastoma cells, providing a better understanding of their subsequent progression and metastasis mechanisms in nude mice. Micrometastases in the lung were initiated from clusters of 2-6 cells rather than single cells in most cases; tumor cells were also visualized binding to the endothelium of small blood vessels to initiate these micrometastases. Shortterm, these mechanisms relied heavily on fluidity of cell surface proteins, rather than nuclear events. Micrometastases in some organs were transient and never became established. Angiogenesis was visualized in both primary tumor systems via "fixation" of the animal's circulation; very small microvessels were growing toward the primary tumor as soon as 48-72 hours post-injection. Marker genes were also valuable for quantitating genetic instability of specific tumor cell populations and potential gene regulatory mechanisms operating in specific organ sites. These latter studies have direct relevance to the significance of N-myc oncogene amplification in neuroblastoma during progression and CD44 gene plasticity of expression in fibrosarcoma during metastasis. Marker gene-tagged single tumor cells can now be analyzed for gene regulatory events in virtually any organ and in combination with laser capture microdissection and other high-resolution methodologies, providing insight into the very earliest gene-regulatory events during micrometastasis.

Cell type-specific modulation of fibronectin adhesion functions on chemically-derivatized self-assembled monolayers.

Cell type-specific responses (microfilament stress fibers for fibroblasts or neurites for neuroblastoma cells) were evaluated in culture on inert and chemically-derivatized silane substrata adsorbed with fibronectin (Fn). Substrata of self-assembled monolayers contain a 14-17 carbon aliphatic chain terminating with different chemical endgroups -- [CH3], [C=C], [Br], [CN], [Diol], [COOH], [NH2], [SH], [SCOCH3], or [SO3H]. Fn adsorbed effectively to all derivatized surfaces. 3T3 fibroblasts or neuroblastoma cells attached equivalently to all surfaces preadsorbed with Fn, indicating availability of receptor binding sites on Fns. However, transmembrane signaling from Fn(adsorbed): receptor(cell) surface complexes yielded a range of abilities for generating F-actin stress fibers in fibroblasts or neurites in neuroblastoma cells. Efficiency for stress fiber formation was very different from that of neurite extension. The same chemical endgroups on glass, titanium, or germanium yielded the same patterns of cellular physiological responses, indicating that inert substrata do not act at a distance and that only chemical endgroups regulate Fn signaling functions. When adhesion-inert albumin is co-adsorbed with Fn, efficiency of neurite extension is improved on some surfaces or diminished on others. These results indicate that the conformation of Fn(adsorbed) changes in specific ways on derivatized substrata. Change in Fn conformation was confirmed by FTIR/ATR spectroscopy experiments of Fn(adsorbed). Overall, these studies indicate changes in Fn conformations on chemically-derivatized self-assembled monolayers leading to up- or down-regulation of cell type-specific physiological responses from receptors via their signaling pathways. They also offer predictability for regulating responses of specific cell types when these cells interact with biomaterial implants in vivo.

Plasticity of CD44s expression during progression and metastasis of fibrosarcoma in an animal model system.

CD44s (standard isoform), which binds hyaluronan (HA), has been analyzed for its significance during fibrosarcoma progression and metastasis in an athymic nude mouse model using Balb/c 3T3 cells transformed with ras or sis oncogenes. While ras (but not sis) transformation leads to upregulated expression of mouse CD44s and HA binding, transfection/overexpression of human CD44s gene (hCD44s) elicited remarkable plasticity of expression during progression and metastasis. In 3T3, hCD44s induced tumorigenesis, HA binding, and micrometastatic competence to lungs and other organs. In tumorigenic (but nonmetastatic) sis transformants or ras-deleted revertants, it also induced micrometastatic competence. Conversely, large primary tumors and overt metastases lost hCD44s expression and HA binding via hypermethylation of hCD44s gene. Tail vein injections revealed that hCD44s greatly increased the efficiency of colonization of the lung microvasculature at the earliest stages. These studies indicate that hCD44s overexpression and possibly its HA binding are critical for conveying metastatic competence but are antagonistic or selected against during aggressive primary tumor or overt metastasis outgrowth. This remarkable plasticity of expression and its consequences offer an ideal system for dissecting the molecular mechanisms operating during fibrosarcoma progression and metastasis.

Earliest steps in primary tumor formation and micrometastasis resolved with histochemical markers of gene-tagged tumor cells.

To facilitate detection of tumor cells at the highest resolution in any organ in athymic nude mouse model systems, a histochemical marker gene [bacterial lacZ or human placental alkaline phosphatase (ALP)] was transfected into specified transformed/tumor cells (fibrosarcoma or neuroblastoma). The fates of tumor cells were followed qualitatively and quantitatively by histochemical staining of whole organs or organ sections. Primary tumors developed initially via formation of "curly-haired" complexes of cells in the subcutis or dermis, followed by division of a large fraction of cells. When two tumor classes were mixed before injection, outgrowth occurred in regional concentrations of the primary tumor. Blood microvessels were detectable within 72 hr of injection, growing into tumor regions. iv injection routinely yielded multicellular foci in the lungs within minutes as precursors of experimental metastases. Micrometastasis was further resolved with cells "inactivated" by different treatments and by co-injection of two different tagged cell types. These approaches using different histochemical marker genes to "tag" different tumor cell classes, along with more advanced molecular biological approaches, permit us to characterize gene expression and its reversibility during the earliest stages of primary tumor formation and micrometastasis to virtually any organ in the recipient animal.

Adhesion to fibronectin's EDb domain induces tyrosine phosphorylation of focal adhesion proteins in Balb/c 3T3 cells.

Balb/c 3T3 cell adhesion on substrata coated with fibronectin's (FN) alternatively-spliced EDb, implicated in some tumor cell systems, and its neighboring type III repeats (III7 and III8) induced intracellular signaling coincident with morphological responses. These events were analysed using minigene-expressed proteins containing various permutations of type III repeats of FN. Cells adherent to the tri-repeat protein 7-EDb-8 were compared to those attached to the tri-repeat 8-9-10 which can interact with integrins through RGD and its synergistic sequences. Cell adhesion to 7-EDb-8 generated rapid tyrosine phosphorylation of several intracellular proteins (particularly the complex at 120-130 kD), with the overall phosphorylation level and its sensitivity to tyrosine kinase inhibitors similar to responses on the 8-9-10 tri-repeat. This similarity contrasted with the differential morphological responses of cells mediated by these proteins. Further analysis of the kinetics of phosphorylation through immunoblotting of two focal adhesion proteins, p125FAK and pl30cas, revealed a profile for Balb/c 3T3 adhesion to 7-EDb-8 distinct from that on 8-9-10. EDb mono-repeat was also more potent for inducing both limited cell spreading and FAK tyrosine phosphorylation than its neighboring repeats III7 or III8. Examination of cellular localization of FAK and vinculin showed that cells spread on the 7-EDb-8 substratum displayed vinculin-positive focal complex-like structures at the cell periphery, in contrast to the classical focal adhesions seen in 8-9-10-adherent cells. These results suggest that EDb induces cell signaling events, leading to tyrosine phosphorylation of focal adhesion proteins, through a mechanism different from that mediated by integrins recognizing sequences in III8-9-10. EDb-dependent signaling may have special significance in some tumor systems.

Over-expression of human CD44s in murine 3T3 cells: selection against during primary tumorigenesis and selection for during micrometastasis.

Human CD44 standard isoform (hCD44s) cDNA regulated by a high-expressing promoter was transfected into Balb/c 3T3 cells and the tumorigenic and metastatic capacities of the transfectants were investigated in nude mice at the subcutaneous site. One of three transfectants was tumorigenic. hCD44s expression was lost in the cells of large primary tumors using this tumorigenic clone. These tumors were extremely aggressive giving overt metastases and micrometastases to several sites including mesentery, stomach, liver, diaphragm, pancreas and lung. Micrometastatic cells re-expressed hCD44s, consistent with its importance for early steps in the metastatic cascade. hCD44s was not expressed in overt metastases; most probably the expression was lost during the outgrowth of micrometastases into overt metastatic tumors. Thus hCD44s expression in murine 3T3 cells does induce tumorigenicity in select cases, is not compatible with aggressive outgrowth of primary or secondary tumors, and is advantageous for early steps in metastatic spread. These results suggest that CD44s is an example of a novel type of 'metastasis' molecule that is disadvantageous for tumor growth and is only transiently advantageous during metastatic spreading of tumor cells to distant organs.

Tumor progression, micrometastasis, and genetic instability tracked with histochemical marker genes.

Mouse fibrosarcoma (3T3 cells transfected with different oncogenes), human neuroblastoma, or human prostate carcinoma cells have been genetically-tagged with different histochemical marker genes (E. coli lacZ, placental alkaline phosphatase, or Drosophila alcohol dehydrogenase). Injection into athymic nude mice permits their tracking at all stages of primary tumor formation and micrometastasis to various organs at the single-cell level. Two different tumor classes, tagged with different marker genes, can be tracked together. Primary tumors display regional dominance of one tumor class with exclusion of other classes. During micrometastasis, tumor cells are detected binding to the endothelium of lung blood vessels, followed by establishment of multiple-cell micrometastases. Micrometastases in some organs are transient while in other organs there is differential expansion into overt metastases. Tagged tumors also reveal the timing of angiogenesis of developing primary tumors and overt metastases. In all three tumor systems, there are three classes of genetic stability of marker gene expression in clonal populations-high stability, intermediate stability, and high instability. Instability in marker gene expression in one tagged prostate carcinoma system does not depend on a hypermethylation mechanism, suggesting a genetic basis for loss of activity. Use of histochemical marker genes, combined with laser-capture microdissection and various PCR methods, can now be used to evaluate gene activities in single or multiple tumor cells in virtually any organ and primary tumor of the animal model system.

Overexpressed human CD44s promotes lung colonization during micrometastasis of murine fibrosarcoma cells: facilitated retention in the lung vasculature.

Normally nonmetastatic murine sis-transformed BALB/c 3T3 cells, transfected with human CD44s gene (hCD44s), acquire spontaneous metastatic capacity to the lung. The mechanism(s) of this facilitated micrometastasis was analyzed in an experimental metastasis model. Human CD44s overexpression promoted the earliest stages severalfold (initial implantation and subsequent stabilization of tumor cells) but was irrelevant for later stages (subsequent outgrowth) of lung experimental micrometastasis. By injecting mixed populations of parental (nonmetastatic) and CD44s-transfected cells, it was shown that cell-cell adhesion between tumor and parental cells was not promoted by hCD44s but that promotion of cell-cell adhesion to lung endothelium or specifically between transfected cells (via hyaluronan) are likely mechanisms. Results obtained with hCD44s-negative primary tumor cells and hCD44s-positive or -negative variants of lung micrometastatic cells (after s.c. injection of transfectants) confirmed the importance of CD44s overexpression for early but not late stages of experimental lung metastasis. Therefore, CD44s represents a metastasis-facilitating molecule that is irrelevant for primary tumor outgrowth but that promotes micrometastasis to the lungs at the very earliest stages.

Counter-selection for over-expressed human CD44s in primary tumors versus lung metastases in a mouse fibrosarcoma model.

Human CD44 standard isoform cDNA (hCD44s) was transfected into sis-transformed Balb/c 3T3 cells and into ras-revertant IIIA4 cells (both tumorigenic but nonmetastatic). Transfectants were injected subcutaneously into athymic nude mice to elucidate the functional role of hCD44s over-expression in progression and metastasis. The transfectants (but not parental cells) were capable of lung micrometastasis and of binding exogenously-added hyaluronan. hCD44s protein expression was conserved in lung micrometastases suggesting that it may have been necessary for their formation. In contrast, no hCD44s protein was detected in large subcutaneous (s.c.) tumors but normal levels of murine CD44 were detected. A second round of tumor development, using these two tumor cell classes, demonstrated that hCD44s-nonexpressing s.c. tumor cells re-expressed it in lung micrometastases. Conversely, hCD44s-expressing lung micrometastatic cells, when injected into a second group of mice, downregulated hCD44s expression in order to grow sizable s.c. tumors. S.c. tumor cells still contained the hCD44s gene but its expression was inhibited by epigenetic mechanisms, one of which was shown to be methylation of the hCD44s gene. These studies demonstrate (a) opposing selective pressures on CD44s over-expression for s.c. tumor growth and for metastatic spread to the lung and (b) further credence for the significance of CD44 for metastatic spread of fibrosarcomas. Therefore, CD44s may be a critical component of the metastatic phenotype induced by specific oncogenes.

N-myc over-expression downregulates alpha3beta1 integrin expression in human Saos-2 osteosarcoma cells.

Alterations in adhesion to the extracellular matrix mediated by integrin receptors are commonly observed in a wide variety of transformed/tumor classes. Reductions in the expression of several integrin subunits have been documented in human neuroblastoma cell lines that over-express the neuroblastoma-associated oncogene N-myc. Neuroblastoma cells transfected with a cDNA encoding N-myc on a high-expression plasmid exhibit greatly reduced levels of alpha2, alpha3 and beta1 integrin subunits with concomitant rounding of cells on substrata. In the current studies, we examined whether integrin downregulation by N-myc is cell-type specific by transfecting a human N-myc cDNA into Saos-2 human osteosarcoma cells and evaluating integrin expression. Several N-myc-expressing cell lines were isolated which exhibit reduced levels of beta1 integrin subunit protein and significant alteration in cell morphology - these cell lines resemble N-myc-over-expressing neuroblastoma cells. In addition to reduced beta1 subunit levels, the osteosarcoma-derived N-myc transfectants exhibit little or no alpha3beta1 integrin complexes, either intracellular or at the cell surface. Finally, reduced amounts of alpha3 integrin subunit in these cell lines occur at the level of alpha3 integrin mRNA, although post-transcriptional mechanisms may also be involved, particularly with inability of pre-beta1 protein to mature. These results confirm our previous studies demonstrating integrin downregulation by an N-myc-dependent process and, in addition, demonstrate lack of cell-type specificity in the action of N-myc on integrin extracellular matrix receptor expression when comparing neural precursor (neuroblastoma) cells with connective tissue (osteosarcoma) cells.

Concomitant down-regulation of expression of integrin subunits by N-myc in human neuroblastoma cells: differential regulation of alpha2, alpha3 and beta1.

Amplification and over-expression of the N-myc oncogene is associated with the progression of neuroblastoma in children and in a nude mouse model system. Neuroblastoma cell lines with widely different levels of N-myc illustrate an inverse relationship between N-myc over-expression and reduced expression of several integrin extracellular matrix receptors. Transfection and over-expression of N-myc in a neuroblastoma cell line not normally expressing the protein resulted in cells that grew loosely associated with tissue culture plates; this correlated with reduced levels of beta1 integrin subunit. Evidence is now presented that alpha2 and alpha3 integrin subunit levels are also reduced in cells that over-express N-myc, with virtually no association of alpha2 or alpha3 subunits with beta1. Consequently, maturation of the beta1 subunit and cell surface expression of the integrins are greatly reduced in N-myc-transfected cells. A small amount of beta1 protein does get to the cell surface, however, suggesting that an as yet unidentified alpha subunit is produced by the N-myc-expressing cells. Finally, the observed reductions in integrin protein levels are reflections of greatly reduced levels of integrin alpha2 and alpha3 mRNAs, as well as a smaller reduction in beta1 mRNA (80%, 94% and 52%, respectively). Post-transcriptional mechanisms modulating beta1 integrin levels are also operative. These results indicate that over-expression of N-myc from a transfected gene in a neuroblastoma cell line that does not normally produce the protein generates cell lines with many of the characteristics of naturally metastatic cells with amplified N-myc genes. Modulation of N-myc and integrin expressions may play a significant role in progression of human neuroblastoma.

CD44 protein levels and its biological activity are regulated in Balb/c 3T3 fibroblasts by serum factors and by transformation with the ras but not with the sis oncogene.

CD44s (standard isoform) levels and hyaluronan-binding activity were investigated in Balb/c 3T3 cells and their derivatives transformed with ras or sis oncogenes as a function of serum concentration in the medium. 3T3 cells contained low levels of CD44 and did not bind hyaluronan when grown in medium containing 0.5 or 10% serum. In 5% serum, however, the cells had much higher levels of CD44 and were able to bind hyaluronan. CD44 levels also increased in 3T3 cells restimulated with either 5 or 10% serum after prior maintenance in low serum. In cells restimulated with 5% serum, high levels of CD44 were sustained for at least 72 hr. In cells restimulated with 10% serum, however, the increase in CD44 levels reverted by 48 hr. Transformation of 3T3 cells with ras (but not with sis) oncogene rendered CD44 levels insensitive to serum modulation: ras-transformed cells contained high levels of CD44 and bound hyaluronan at all serum concentrations and at all time points tested. Sis-transformed cells behaved like 3T3 cells in these modulatory changes. Platelet-derived growth factor (PDGF), when supplementing 0.5% serum, mimicked the effects of serum on the levels and hyaluronan-binding capacity of CD44 in 3T3 cells and the CD44-upregulating activity of serum was neutralized by incubation with anti-PDGF antibodies. These data demonstrate that serum factors, specifically PDGF, mediate regulation of CD44 levels in BAlb/c 3T3 cells and that transformation of 3T3 cells by ras renders CD44 expression insensitive to the modulating effects of serum in vitro. These results correlate with the metastatic capacity of these cells in vivo.

Induction of monocyte expression of tumor necrosis factor alpha by the 30-kD alpha antigen of Mycobacterium tuberculosis and synergism with fibronectin.

Native 30-kD antigen, also known as alpha antigen, is a fibronectin-binding protein that is secreted by live Mycobacterium tuberculosis. This antigen may play an important biological role in the host-parasite interaction since it elicits delayed type hypersensitivity response and protective immunity in vivo and T lymphocyte blastogenesis and IFN-gamma production in vitro. In the present study, we show that, TNF-alpha protein is produced in monocyte culture supernatants in response to 30-kD antigen and the level is as high as that to purified protein derivative of M. tuberculosis. This stimulatory effect was not due to contamination with either bacterial lipopolysaccharide or mycobacterial lipoarabinomannan. The preincubation of monocytes with plasma fibronectin significantly enhanced the release of TNF-alpha into the culture supernatants in response to 30-kD antigen. This effect was blocked by polygonal antibody to plasma fibronectin. In contrast, the monocytic cell line U937 failed to release TNF-alpha protein in the culture supernatants in response to 30-kD antigen with or without preincubation with plasma fibronectin. To determine whether this observation was due to differential binding of the 30-kD to fibronectin on these cells, a cell based ELISA was used. Pretreatment of monocytes with fibronectin enhanced their binding of the 30-kD antigen. U937 cells bound the 30-kD antigen weakly with or without fibronectin pretreatment. These results indicate that 30-kD antigen which is a known secretary antigen of M. tuberculosis is a stimulus for human monocytes to express TNF-alpha and that stimulatory effect may be mediated through plasma fibronectin.

Adhesion mediated by fibronectin's alternatively spliced EDb (EIIIB) and its neighboring type III repeats.

Adhesion functions of cellular fibronectin's (FN) alternatively spliced EDb (EIIIB) domain, as well as its neighboring type III repeats III7 and III8, were investigated with several cultured murine and human cell types. Minigene constructs encoding various permutations of these repeats and expressed in bacteria were used as shown previously in function studies of EDa and its neighboring repeats (P.Xia and L. A. Culp Exp. Cell Res. 213, 253-265, 1994). When substrata of recombinant proteins were incubated with several fibroblastic or neuronal derivative cell lines, cell attachment responses varied widely in a cell-type-specific manner. Balb/c 3T3 cells were shown to adhere to recombinant protein substrata in dose-dependent and EDTA-sensitive manners. Responses also varied with which repeat combinations were being tested, from excellent (Balb/c 3T3, src-3T3), to intermediate (Platt cells), to poor (LZEJ, VA-13, and F11), with EDb plus-containing proteins generally giving better adhesion than EDb minus proteins. On select recombinant proteins, cells showed limited cytoplasmic spreading (3T3 and src-3T3) or neurite extension (Platt and F11), while other cell lines (VA-13 and LZEJ) did not show any morphological changes beyond attachment. Again, EDb plus-containing recombinants were more effective at inducing these morphological changes than the neighboring repeats. These results demonstrate that the EDb domain of cellular FNs and its neighboring type III homology repeats contain important adhesion-promoting sequences, which may be regulated by cells through alternative splicing of FN's primary transcript.

Oncogene-dependent expression of CD44 in Balb/c 3T3 derivatives: correlation with metastatic competence.

Oncogene-dependent regulation and tumor relatedness of CD44 expression were investigated in Balb/c 3T3 cells and their derivatives transformed with different ras oncogenes (metastatic tumor model) or the human c-sis oncogene (non-metastatic model). Ras transformants using either the Harvey or Kirsten oncogenes expressed high levels of cell surface CD44 protein that bound fluoresceinated hyaluronan (HA). Much lower levels of CD44 were expressed in parental 3T3 cells, ras- revertants generated from Kirsten-transformed cells, or c-sis transformants, confirming the significance of the ras oncogene in this upregulation. To determine whether endogenous HA regulates these parameters, hyaluronidase treatment of ras transformants exposed more cell surface CD44 to anti-CD44 antibody and increased fluoresceinated HA binding; this did not occur with 3T3 or c-sis transformants. CD44 expression and its HA-binding function were conserved in a panel of in vivo primary and lung metastatic tumor cell lines derived from ras transformants. Ras transformants also retained the ability to downregulate CD44 protein levels in confluent cultures which occurred through a translational or post-translational mechanism (as CD44 mRNA levels were not reduced). These results taken together demonstrate that ras-dependent regulation of CD44 may correlate with tumor progression and metastasis in vivo, possibly (although not exclusively) supporting CD44's importance in metastatic progression.

Over-expression of transfected N-myc oncogene in human SKNSH neuroblastoma cells down-regulates expression of beta 1 integrin subunit.

Amplification of the N-myc oncogene is associated with progression of neuroblastoma in humans. Previous studies indicated that neuroblastoma cell lines which are amplified for the N-myc gene and over-express N-myc exhibit enhanced tumorigenic properties when injected into athymic nude mice. In addition, neuroblastoma cells which over-express N-myc (IMR32 cells) expressed little or no beta 1, alpha 2, or alpha 3 integrin subunits, as compared with cells which do not express N-myc (SKNSH cells). In order to probe the possible relationship between N-myc and beta 1 integrin gene expressions more directly, transfection experiments were performed in which an N-myc cDNA (on the episomal expression vector pREP4; high-level constitutive expression is driven by an RSV-LTR promoter) was introduced into SKNSH cells. Expression of N-myc produced significant morphological alterations in transfected cells; one subpopulation of cells remained spread on tissue culture substrata, while a second subpopulation became rounded and grew as multi-cellular aggregates. Spread (attached) cells expressed low levels of N-myc and high levels of beta 1 integrin, while rounded (loose) cells expressed relatively high levels of N-myc and low levels of beta 1 integrin. Maintenance of transfected cells in higher concentrations of selective agent produced a higher proportion of loose cells, which expressed even greater amounts of N-myc and even less beta 1 integrin; a similar effect was observed in attached cells. Interestingly, loose cell populations expressed elevated levels of the neural cell adhesion molecule [NCAM]. The results presented here infer that N-myc regulates the expression of the beta 1 integrin and NCAM cell-surface receptors responsible for cell:extracellular cellular matrix interaction and possibly cell:cell adhesion.

Adhesion activity in fibronectin's alternatively spliced domain EDa (EIIIA): complementarity to plasma fibronectin functions.

The EDa (EIIIA) domain is one of two alternatively spliced type III homology repeats in fibronectins which differentiate cellular (cFN) from plasma isoforms (pFN). A bacteria-expressed recombinant polypeptide encoding the EDa type III repeat promotes adhesion of some cell types and its activity is synergistic with neighboring repeats III11 and III12. We show in this study that co-coating substrata with the EDa-only polypeptide and a suboptimal concentration of pFN leads to increased attachment and extensive spreading of v-src-transformed 3T3 cells relative to that found on substrata of suboptimal pFN or EDa polypeptide alone. This complementarity of activities requires as little as 1 microgram/ml EDa polypeptide in the adsorbing mixture and displays sequence specificity for only EDa (recombinant polypeptides of neighboring repeats III11 or III12 were without effect). Furthermore, stress fibers and focal contacts are inducible on the EDa:pFN mixture, suggesting that the EDa sequence and its receptor participate in signal transduction. The codistribution of phosphotyrosine proteins, including pp125FAK, along with vinculin and talin into focal contacts supports this hypothesis. Therefore, an alternatively spliced domain EDa which is expressed in various proportions in cells and tissues may have special functions related to adhesion processes by complementing the functions of pFN circulating in blood.

Inverse expressions of the N-myc oncogene and beta 1 integrin in human neuroblastoma: relationships to disease progression in a nude mouse model system.

A nude mouse model for human neuroblastoma has been developed to examine possible relationships between amplification/over-expression of the N-myc oncogene and altered regulation of expression of specific integrin subunits during tumor progression. Subcutaneous (ectopic) or intra-adrenal (orthotopic) injection of the neuroblastoma cell lines SK-N-SH or IMR-32 has generated a number of derivative tumor cell lines. Tumor cell lines derived from SK-N-SH cells (which do not express N-myc) or IMR-32 cells (which over-express N-myc) produce tumors at higher rates when re-injected into the subcutaneous space of nude mice. Moreover, cell lines derived from tumors initiated by IMR-32 cells exhibit shorter latent periods than do IMR-32 cells direct from tissue culture. With regard to integrin subunit expression, SK-N-SH and related cell lines express high levels of beta 1 integrin, which is associated with the alpha 2 and alpha 3 integrin subunits (predominantly alpha 3). IMR-32 cells display reduced beta 1 expression, and that which is produced is not associated with common alpha subunits. LaN1 cells, which express N-myc at even higher levels than do IMR-32 cells, express even less beta 1. Interestingly, the tumor-derived cell lines (especially those from tumors initiated in adrenal glands) also exhibit reduced integrin expression compared with the parental cell lines; this reduction is associated with the enhanced tumor take rate observed when the cells are re-injected into nude mice. Our results raise the possibility of a relationship between over-expression of N-myc and down-regulation of beta 1 integrin expression (possibly some alpha subunits also). In addition, the data suggest that human neuroblastoma-derived cell lines which exhibit reduced integrin expression display more aggressive tumor growth in nude mice.