Novel methods for chemiluminescent detection of reporter enzymes.

Chemiluminescent reporter gene assays provide highly sensitive, quantitative detection in simple, rapid assay formats for detection of reporter enzymes that are widely employed in gene expression studies. Chemiluminescent detection methodologies typically provide up to 100-1000x higher sensitivities than may be achieved with fluorescent or colorimetric enzyme substrates. The variety of chemiluminescent 1,2-dioxetane substrates available enable assay versatility, allowing optimization of assay formats with the available instrumentation, and are ideal for use in gene expression assays performed in both biomedical and pharmaceutical research. In addition, 1,2,-dioxetane chemistries can be multiplexed with luciferase detection reagents for dual detection of multiple enzymes in a single sample. These assays are amenable to automation with a broad range of instrumentation for high throughput compound screening.

Propensity for macrophage apoptosis is related to the pattern of expression and function of integrin extracellular matrix receptors.

Ligation of integrins to an extracellular matrix activates signal transduction systems which produce multiple responses in different cell types. Adhesion often provides a survival signal to cells; disruption of adhesion frequently results in apoptosis. Our laboratory has utilized apoptosis-sensitive and -resistant cell lines to investigate the role of integrin expression and function in regulation of apoptosis in macrophages. Chronic exposure of murine macrophage-like RAW264.7 cells to apoptosis-inducing agents (bacterial lipopolysaccharide and interferon-gamma) resulted in the generation of a derivative cell line (RES) resistant to apoptosis. Observation of RAW and RES cultures indicated a difference in adhesion between the two cell types. The two cell lines also exhibit significant differences in expression of integrins previously characterized to be important in apoptosis.

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.

Cellular adaptive responses to low oxygen tension: apoptosis and resistance.

Oxygen plays such a critical role in the central nervous system that a specialized mechanism of oxygen delivery to neurons is required. Reduced oxygen tension, or hypoxia, may have severe detrimental effects on neuronal cells. Several studies suggest that hypoxia can induce cellular adaptive responses that overcome apoptotic signals in order to minimize hypoxic injury or damage. Adaptive responses of neuronal cells to hypoxia may involve activation of various ion channels, as well as induction of specific gene expression. For example, ATP sensitive K+ channels are activated by hypoxia in selective neuronal cells, and may play a role in cell survival during hypoxia/anoxia. Additionally, hypoxia-induced c-Jun, bFGF and NGF expression appear to be associated with prevention (or delay) of neuronal cell apoptosis. In this paper, these adaptive responses to hypoxia in neuronal cells are discussed to examine the possible role of hypoxia in pathophysiology of diseases.

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.

Inflammatory mediators are perpetuated in macrophages resistant to apoptosis induced by hypoxia.

A hypoxic/anoxic microenvironment has been proposed to exist within a vascular lesion due to intimal or medial cell proliferation in vascular diseases. Here, we examined whether hypoxia alters macrophage function by exposing murine macrophage-like RAW 264.7 (RAW) cells to hypoxia (2% O2). When cells were exposed to hypoxia, a significant number of RAW cells underwent apoptosis. Additionally, small subpopulations of RAW cells were resistant to hypoxia-induced apoptosis. Through repeated cycles of hypoxia exposure, hypoxia-induced apoptosis-resistant macrophages (HARMs) were selected; HARM cells demonstrate >70% resistance to hypoxia-induced apoptosis, as compared with the parental RAW cells. When heat shock protein (HSP) expression was examined after hypoxia, we observed a significant decrease in constitutive heat shock protein 70 (HSC 70) in RAW cells, but not in HARMs, as compared with the control normoxic condition (21% O2). In contrast, the expression level of glucose-regulated protein 78 (GRP 78) in RAW and HARM cells after hypoxia treatment was not altered, suggesting that HSC 70 and not GRP 78 may play a role in protection against hypoxia-induced apoptosis. When tumor necrosis factor alpha (TNF-alpha) production was examined after hypoxic treatment, a significant increase in TNF-alpha production in HARM but decrease in RAW was observed, as compared with cells cultured in normoxic conditions. HARM cells also exhibit a much lower level of modified-LDL uptake than do RAW cells, suggesting that HARMs may not transform into foam cells. These results suggest that a selective population of macrophages may adapt to potentially pathological hypoxic conditions by overcoming the apoptotic signal.

Persistent alpha 1 integrin subunit expression in human neuroblastoma cell lines which overexpress N-myc and downregulate other integrin subunits.

Neuroblastoma is characterized by amplification and overexpression of N-myc. N-myc down-regulates expression of the beta 1 integrin extracellular matrix receptor, however, some beta 1 was found on the surface of N-myc overexpressing neuroblastoma cell lines. It was determined that the alpha 1 subunit is expressed in the cells, associates with beta 1 and is present on the cell surface. Interestingly, the level of alpha 1 was reduced marginally when compared to beta 1. Finally, overexpression of N-myc alters cell morphology on extracellular matrix proteins with persistence of alpha 1 beta 1-dependent responses. Our work supports the conclusion that altered integrin expression may be an important factor in human neuroblastoma.

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.

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.

Integrin expression in human neuroblastoma cells with or without N-myc amplification and in ectopic/orthotopic nude mouse tumors.

Three human neuroblastoma cell lines, with or without N-myc amplification, were evaluated for their integrin expression patterns as cultured cells, as well as their nude mouse-borne tumors obtained after subcutaneous (ectopic) or adrenal gland (orthotopic) injection. IMR-32 and LaN1 cells (with amplified N-myc) do not express any of the common integrin subunits that recognize fibronectin or collagens, as determined by immunoprecipitation of cell extracts with specific monoclonal antibodies; the same was true for all subcutaneous or adrenal tumors from IMR-32 or LaN1, indicating that they are not essential during primary tumor formation at either site. SK-N-SH cells (with diploid N-myc) express beta 1, alpha 2, and alpha 3 subunits of expected sizes (with alpha 2 uncleaved at 145 kDa) but do not express alpha 1, alpha 4, alpha 5, alpha V, or beta 3. This expression pattern was conserved in all first-round subcutaneous and adrenal tumor cell populations, as well as in second-round subcutaneous tumors derived from a first-round subcutaneous tumor (no tumors expressed beta 3). One significant difference was noted between subcutaneous and adrenal tumor populations: all first- and second-round subcutaneous tumors expressed high levels of alpha V subunit, while adrenal tumors did not express any alpha V. This result suggests some essential function for alpha V beta 1 during subcutaneous primary tumor formation. Integrin patterns were also evaluated by fluorescence-activated cell sorting. SK-N-SH and its derivative tumors expressed heterogeneous amounts of beta 1 and alpha 2 at the cell surface, while only subcutaneous tumor cells expressed alpha V. Parental SK-N-SH cells contained two subpopulations, half of which expresses alpha 3, while the other half does not; all subcutaneous tumor cells retained this two-subpopulation pattern, indicating that primary tumor formation does not lead to clonal dominance of alpha 3- or alpha 3+ cell types in larger primary tumors. While these results suggest a correlation between N-myc amplification and down-regulation of integrin expression in neuroblastoma, they demonstrate conservation of integrin expression during two rounds of primary tumor formation at ectopic or orthotopic sites in a mouse model system, induction and/or selection for alpha V beta 1 expression at the subcutaneous site, and clonal heterogeneity in alpha 3 beta 1 expression throughout primary tumor development.

Inhibition of the dsRNA-dependent protein kinase by a peptide derived from the human immunodeficiency virus type 1 Tat protein.

The human immunodeficiency virus (HIV) is the etiologic agent leading to the development of acquired immunodeficiency syndrome (AIDS). Interferons (IFNs) are known for eliciting antiviral responses from cells, and studies have indicated that infection with HIV induces the production of IFN. Previous studies have shown that the trans-acting response element (TAR) sequence of HIV-1 mRNA can activate the IFN-induced double-stranded (ds) RNA-dependent protein kinase (DAI). DAI, when activated, is a potent inhibitor of protein synthesis and has been implicated in mediating part of IFN's antiviral activity. Here, we report that a synthetic peptide containing the basic region of HIV Tat protein is effective in preventing the activation of DAI. Evidence is presented that indicates that the Tat peptide exerts its effect by binding to the TAR RNA sequence and thus preventing this RNA from binding to and activating DAI. It appears that in addition to its role in trans-activation, the tat protein may also function to overcome the antiviral activity of IFN by regulating DAI activity. Thus, inhibition of DAI by the Tat protein early in the life cycle of HIV may provide a mechanism by which the virus can escape a translational block imposed by the kinase.

Mechanism of action of a cellular inhibitor of the dsRNA-dependent protein kinase from 3T3-F442A cells.

When mouse 3T3-F442A preadipocyte fibroblasts reach confluence in the appropriate culture medium, their growth is arrested, and the cells undergo terminal differentiation to adipocytes. Two proteins that may be involved in this process are interferon and the interferon-induced double-stranded RNA (dsRNA)-dependent protein kinase (DAI). In 3T3-F442A cells, interferon and DAI are transiently expressed with a maximum level of active kinase appearing at confluence. Interestingly, the level of active DAI was found to be low when cells were maintained under conditions nonpermissive for differentiation. This reduction in DAI was at least partly because of the presence of elevated levels of a specific inhibitor of DAI, termed dRF, which appeared to be a reversible inhibitor of the autophosphorylation (activation) of DAI. In the present study, the mechanism of action of dRF was investigated. Photocross-linking experiments indicated that dRF prevented the binding of ATP to DAI. Since the binding of ATP to DAI is dsRNA-dependent, we examined the effect of dRF on the binding of dsRNA to the kinase using RNA mobility shift assays. dRF was found to prevent the formation of DAI-dsRNA complexes without a direct effect on the dsRNA. This suggests that dRF exerts its effect through an interaction with DAI.

Partial characterization of a cellular factor that regulates the double-stranded RNA-dependent eIF-2 alpha kinase in 3T3-F442A fibroblasts.

The interferon-induced double-stranded RNA-dependent eIF-2 alpha kinase (dsI) has an established role in mediating part of interferon's antiviral effects. Numerous studies have suggested that dsI also has regulatory functions in cells not infected with virus. Our previous results have indicated that the activation of this kinase may be an important regulatory signal in controlling growth arrest of mouse 3T3-F442A fibroblasts prior to their subsequent differentiation to adipocytes. Here, we report that extracts from 3T3-F442A cells cultured under conditions nonpermissive for differentiation exhibit significantly reduced dsI activity and that this reduction is due, at least in part, to the presence of elevated levels of a novel inhibitor of dsI activation (dRF). This inhibitor is also detected in reduced amounts in extracts from cells cultured under conditions which are permissive for differentiation. We have achieved a 1,000-fold purification of dRF activity, and highly purified dRF preparations were found to be greatly enriched for a 15-kDa protein that was greater than 90% pure. Our results indicate that dRF is not a protein phosphatase or protease but a reversible inhibitor of dsI autophosphorylation. In addition, our results imply that dRF is a physiologic regulator of dsI, since dRF activity correlates with the ability of 3T3-F442A cells to undergo adipose conversion.