Genomic and metabolomic analysis of step-wise malignant transformation in human skin fibroblasts.

Metabolic changes accompanying a step-wise malignant transformation was investigated using a syngeneic lineage of human fibroblasts. Cell immortalization was associated with minor alterations in metabolism. Consecutive loss of cell cycle inhibition in immortalized cells resulted in increased levels of oxidative phosphorylation (OXPHOS). Overexpression of the H-Ras oncoprotein produced cells forming sarcomas in athymic mice. These transformed cells exhibited increased glucose consumption, glycolysis and a further increase in OXPHOS. Because of the markedly increased OXPHOS in transformed cells, the impact of a transaminase inhibitor, aminooxyacetic acid (AOA), which decreases glutamine influx to the tricarboxylic acid (TCA) cycle, was tested. Indeed, AOA significantly decreased proliferation of malignantly transformed fibroblasts and fibrosarcoma-derived cells in vitro and in vivo. AOA also decreased proliferation of cells susceptible to malignant transformation. Metabolomic studies in normal and transformed cells indicated that, in addition to the anticipated effect on the TCA cycle, AOA decreased production of nucleotides adenosine triphosphate (ATP) and uridine monophosphate. Exogenous nucleotides partially rescued decreased proliferation of the malignant cells treated with AOA. Our data indicate that AOA blocks several metabolic pathways essential for growth of malignant cells. Therefore, OXPHOS may provide important therapeutic targets for treatment of sarcoma.

Rev7/Mad2B plays a critical role in the assembly of a functional mitotic spindle.

The spindle assembly checkpoint (SAC) acts as a guardian against cellular threats that may lead to chromosomal missegregation and aneuploidy. Mad2, an anaphase-promoting complex/cyclosome-Cdc20 (APC/C(Cdc20)) inhibitor, has an additional homolog in mammals known as Mad2B, Mad2L2 or Rev7. Apart from its role in Polζ-mediated translesion DNA synthesis and double-strand break repair, Rev7 is also believed to inhibit APC/C by negatively regulating Cdh1. Here we report yet another function of Rev7 in cultured human cells. Rev7, as predicted earlier, is involved in the formation of a functional spindle and maintenance of chromosome segregation. In the absence of Rev7, cells tend to arrest in G2/M-phase and display increased monoastral and abnormal spindles with misaligned chromosomes. Furthermore, Rev7-depleted cells show Mad2 localization at the kinetochores of metaphase cells, an indicator of activated SAC, coupled with increased levels of Cyclin B1, an APC(Cdc20) substrate. Surprisingly unlike Mad2, depletion of Rev7 in several cultured human cell lines did not compromise SAC activity. Our data therefore suggest that besides its role in APC/C(Cdh1) inhibition, Rev7 is also required for mitotic spindle organization and faithful chromosome segregation most probably through its physical interaction with RAN.

Assessing the in vivo efficacy of doxorubicin loaded hyaluronan nanoparticles.

Magnetic nanoparticles are attractive platforms for biomedical applications including diagnosis and treatment of diseases. We have shown previously that hyaluronan-coated superparamagnetic iron oxide nanoparticles (HA-SPIONs) enhanced the efficacy of the conjugated anticancer drug doxorubicin (DOX) in vitro against drug-sensitive and drug-resistant human ovarian cancer cells. In this manuscript, we report our findings on the efficacy of DOX loaded HA-SPIONs in vivo using subcutaneous and intraperitoneal SKOV-3 ovarian tumor models in nude mice. The accumulation of the nanoparticles in subcutaneous tumors following an intravenous nanoparticle administration was confirmed by magnetic resonance imaging, and its distribution in the tumors was evaluated by confocal microscopy and Prussian blue staining. DOX delivered by nanoparticles accumulated at much higher levels and distributed wider in the tumor tissue than intravenously injected free DOX, leading to significant reduction of tumor growth. The IVIS Spectrum for in vivo bioluminescence imaging was used to aid in therapy assessment of the DOX-loaded nanoparticles on intraperitoneal ovarian tumors formed by firefly luciferase expressing human ovarian SKOV-3 cells. DOX-loaded HA-SPIONs significantly reduced tumor growth, delayed tumor development, and extended the survival of mice. Thus, utilizing HA-SPIONs as drug delivery vehicles constitutes a promising approach to tackle CD44 expressing ovarian cancer.

Exploiting identifiability and intergene correlation for improved detection of differential expression.

Accurate differential analysis of microarray data strongly depends on effective treatment of intergene correlation. Such dependence is ordinarily accounted for in terms of its effect on significance cutoffs. In this paper, it is shown that correlation can, in fact, be exploited to share information across tests and reorder expression differentials for increased statistical power, regardless of the threshold. Significantly improved differential analysis is the result of two simple measures: (i) adjusting test statistics to exploit information from identifiable genes (the large subset of genes represented on a microarray that can be classified a priori as nondifferential with very high confidence], but (ii) doing so in a way that accounts for linear dependencies among identifiable and nonidentifiable genes. A method is developed that builds upon the widely used two-sample t-statistic approach and uses analysis in Hilbert space to decompose the nonidentified gene vector into two components that are correlated and uncorrelated with the identified set. In the application to data derived from a widely studied prostate cancer database, the proposed method outperforms some of the most highly regarded approaches published to date. Algorithms in MATLAB and in R are available for public download.

Reprint of BMCL_19101.

The pathogenesis of rheumatoid arthritis is mainly driven by NF-κB-mediated production of cytokines, such as TNF-α. We report herein that the orally available imidazoline-based NF-κB inhibitor, TCH-013, was found to significantly reduce TNF-α signaling and attenuate collagen antibody induced arthritis in BALB/c mice.

Attenuation of collagen-induced arthritis by orally available imidazoline-based NF-κB inhibitors.

The pathogenesis of rheumatoid arthritis is mainly driven by NF-κB-mediated production of cytokines, such as TNF-α. We report herein that the orally available imidazoline-based NF-κB inhibitor, TCH-013, was found to significantly reduce TNF-α signaling and attenuate collagen antibody induced arthritis in BALB/c mice.

Nonlinear dependence in the discovery of differentially expressed genes.

Microarray data are used to determine which genes are active in response to a changing cell environment. Genes are "discovered" when they are significantly differentially expressed in the microarray data collected under the differing conditions. In one prevalent approach, all genes are assumed to satisfy a null hypothesis, ℍ 0, of no difference in expression. A false discovery (type 1 error) occurs when ℍ 0 is incorrectly rejected. The quality of a detection algorithm is assessed by estimating its number of false discoveries, 𝔉. Work involving the second-moment modeling of the z-value histogram (representing gene expression differentials) has shown significantly deleterious effects of intergene expression correlation on the estimate of 𝔉. This paper suggests that nonlinear dependencies could likewise be important. With an applied emphasis, this paper extends the "moment framework" by including third-moment skewness corrections in an estimator of 𝔉. This estimator combines observed correlation (corrected for sampling fluctuations) with the information from easily identifiable null cases. Nonlinear-dependence modeling reduces the estimation error relative to that of linear estimation. Third-moment calculations involve empirical densities of 3 × 3 covariance matrices estimated using very few samples. The principle of entropy maximization is employed to connect estimated moments to 𝔉 inference. Model results are tested with BRCA and HIV data sets and with carefully constructed simulations.

Malignant transformation of human skin fibroblasts by two alternative pathways.

We developed a telomerase-positive, infinite life span human fibroblast cell strain (MSU-1.0) by transfection of a v-MYC oncogene and spontaneous over-expression of transcription factors SP1/SP3. Loss of expression of p14(ALT) and enhanced expression of SPRY2 gave rise to the MSU-1.1 cell strain. Unlike MSU-1.0 cells, the MSU-1.1 cells can be malignantly transformed by expression of N-RAS(LYS61) or H-Ras(v12) oncoproteins (driven by their original promoters) and expression of a SRC-family protein, v-FES. MSU-1.1 cells can also be malignantly transformed by high expression of these RAS oncogenes or the v-K-RAS oncogene. PDGF-B transformed MSU-1.1 cells give rise to benign tumors (fibromas) in athymic mice. A second route to malignant transformation of the MSU-1.1 cells involves loss of functional TP53 protein by carcinogen treatment and loss of expression of wild type p16(INK). These studies indicate 6-8 "hits" are required to activate the oncogenes and inactivate the suppressor genes we identified.

The role of hRev7, the accessory subunit of hPolζ, in translesion synthesis past DNA damage induced by benzo[a]pyrene diol epoxide (BPDE).

BACKGROUND: DNA polymerase zeta (Polζ) is a specialized DNA polymerase that, unlike classical replicative polymerases, is capable of replicating past DNA lesions, i.e. of performing translesion synthesis (TLS). The catalytic subunit of hPolζ, hRev3, has been shown to play a critical role in DNA damage-induced mutagenesis in human cells, but less is known about the role of hRev7, the accessory subunit of hPolζ, in such mutagenesis. To address this question, we recently generated human fibroblasts with very significantly reduced levels of hRev7 protein and demonstrated that hRev7 is required to protect cells from ultraviolet(254 nm) (UV) radiation-induced cytotoxicity and mutagenesis (McNally et al., DNA Repair 7 (2008) 597-604). The goal of the present study was to determine whether hRev7 is similarly involved in the tolerance of DNA damage induced by benzo[a]pyrene diol epoxide (BPDE), the reactive form of the widespread environmental carcinogen benzo[a]pyrene. METHODS: To determine whether hRev7 also plays a role in protecting human cells from the cytotoxicity and mutagenesis induced by benzo[a]pyrene diol epoxide (BPDE), cell strains with reduced hRev7 were compared to their parental strain and a vector control strain for the effect of BPDE on cell survival, induction of mutations, and the ability to progress through the cell cycle. RESULTS: The results show that cell strains with reduced hRev7 are more sensitive to the cytotoxic effect of BPDE than the control strains, and progress through S-phase at a slower rate than the control cells following BPDE treatment, indicating that hRev7, and likely hPolζ, is required for efficient bypass of BPDE-induced DNA lesions. However, neither the frequency nor kinds of mutations induced by BPDE in cells with reduced hRev7 differ significantly from those induced in the control strains, suggesting that hPolζ is not essential for inserting nucleotides opposite BPDE-induced DNA damage. CONCLUSIONS: Taken together, our results which show that hRev7 is required for TLS past BPDE-induced DNA lesions but that it is not essential for inserting nucleotides opposite such lesions suggest a role for hPolζ in the extension step of translesion synthesis.

Transcriptional activity analysis of promoter region of human PAX9 gene under dexamethasone, retinoic acid, and ergocalciferol treatment in MCF-7 and MDPC23.

PAX9 gene is a member of the family homeobox of transcription factors and performs important function in development and organogenesis. Mutations in PAX9 coding sequences have been implicated in autosomal dominant oligodontia affecting predominantly permanent molars and second premolars. Previous studies have shown that PAX9 is required for secondary palate development and teratogens have been identified as inducers of a tooth and craniofacial malformations. This work focused on the analysis on the 5'-flanking region of the PAX9 gene studying the influence of retinoic acid, dexamethasone, and vitamin D on the expression of PAX9 by expression constructs that carry the reporter gene luciferase. As results, retinoic acid and dexamethasone showed progressive decrease of PAX9 expression. PAX9-pGL3B1 and PAX9-pGL3B2 promoter was inhibited under the treatment of dexamethasone and ergocalciferol. Retinoic acid and dexamethasone did not alter PAX9-pGL3B3 behavior indicating that sequences present between -1106 and +92 were important for the transcriptional activity of PAX9 promoter. In this study, we characterized the transcriptional activity of specific regions of the PAX9 promoter gene and we demonstrated that retinoic acid and ergocalciferol can modulate the transcriptional activity of PAX9 gene.

A new adenovirus based vaccine vector expressing an Eimeria tenella derived TLR agonist improves cellular immune responses to an antigenic target.

BACKGROUND: Adenoviral based vectors remain promising vaccine platforms for use against numerous pathogens, including HIV. Recent vaccine trials utilizing Adenovirus based vaccines expressing HIV antigens confirmed induction of cellular immune responses, but these responses failed to prevent HIV infections in vaccinees. This illustrates the need to develop vaccine formulations capable of generating more potent T-cell responses to HIV antigens, such as HIV-Gag, since robust immune responses to this antigen correlate with improved outcomes in long-term non-progressor HIV infected individuals. METHODOLOGY/PRINCIPAL FINDINGS: In this study we designed a novel vaccine strategy utilizing an Ad-based vector expressing a potent TLR agonist derived from Eimeria tenella as an adjuvant to improve immune responses from a [E1-]Ad-based HIV-Gag vaccine. Our results confirm that expression of rEA elicits significantly increased TLR mediated innate immune responses as measured by the influx of plasma cytokines and chemokines, and activation of innate immune responding cells. Furthermore, our data show that the quantity and quality of HIV-Gag specific CD8(+) and CD8(-) T-cell responses were significantly improved when coupled with rEA expression. These responses also correlated with a significantly increased number of HIV-Gag derived epitopes being recognized by host T cells. Finally, functional assays confirmed that rEA expression significantly improved antigen specific CTL responses, in vivo. Moreover, we show that these improved responses were dependent upon improved TLR pathway interactions. CONCLUSION/SIGNIFICANCE: The data presented in this study illustrate the potential utility of Ad-based vectors expressing TLR agonists to improve clinical outcomes dependent upon induction of robust, antigen specific immune responses.

Rac1 and Cdc42 are regulators of HRasV12-transformation and angiogenic factors in human fibroblasts.

BACKGROUND: The activities of Rac1 and Cdc42 are essential for HRas-induced transformation of rodent fibroblasts. What is more, expression of constitutively activated mutants of Rac1 and/or Cdc42 is sufficient for their malignant transformation. The role for these two Rho GTPases in HRas-mediated transformation of human fibroblasts has not been studied. Here we evaluated the contribution of Rac1 and Cdc42 to maintaining HRas-induced transformation of human fibroblasts, and determined the ability of constitutively activated mutants of Rac1 or Cdc42 to induce malignant transformation of a human fibroblast cell strain. METHODS: Under the control of a tetracycline regulatable promoter, dominant negative mutants of Rac1 and Cdc42 were expressed in a human HRas-transformed, tumor derived fibroblast cell line. These cells were used to determine the roles of Rac1 and/or Cdc42 proteins in maintaining HRas-induced transformed phenotypes. Similarly, constitutively active mutants were expressed in a non-transformed human fibroblast cell strain to evaluate their potential to induce malignant transformation. Affymetrix GeneChip arrays were used for transcriptome analyses, and observed expression differences were subsequently validated using protein assays. RESULTS: Expression of dominant negative Rac1 and/or Cdc42 significantly altered transformed phenotypes of HRas malignantly transformed human fibroblasts. In contrast, expression of constitutively active mutants of Rac1 or Cdc42 was not sufficient to induce malignant transformation. Microarray analysis revealed that the expression of 29 genes was dependent on Rac1 and Cdc42, many of which are known to play a role in cancer. The dependence of two such genes, uPA and VEGF was further validated in both normoxic and hypoxic conditions. CONCLUSION(S): The results presented here indicate that expression of both Rac1 and Cdc42 is necessary for maintaining several transformed phenotypes in oncogenic HRas transformed human cells, including their ability to form tumors in athymic mice. Our data also indicate that expression of either activated Rac1 or Cdc42 alone is not sufficient for malignant transformation of human fibroblasts, although each is required for specific transformed phenotypes. Furthermore, our study elucidates that the expression of several highly significant cancer related genes require the activities of Rac1 and/or Cdc42 which may also play a critical role in cellular transformation.

Sprouty 2 regulates DNA damage-induced apoptosis in Ras-transformed human fibroblasts.

We have reported that expression of Sprouty 2 (Spry2) is necessary for tumor formation by HRas(V12)-transformed fibroblasts. We now report on the role of Spry2 in the inhibition of UV(254 nm) radiation-induced apoptosis in HRas(V12)-transformed human fibroblasts. Silencing Spry2 in this context resulted in increased apoptosis, associated with decreased Akt activation and decreased phosphorylation of HDM2 at Ser-166, which has been shown to stabilize HDM2. As a consequence, when cells with silenced Spry2 were UV-irradiated, they exhibited diminished levels of HDM2 and elevated levels of p53. In agreement with these findings, overexpression of Spry2 in the parental non-transformed fibroblasts led to increased Akt activation and to the stabilization of HDM2. It also led to diminished expression of p53 and decreased apoptosis following UV irradiation. Silencing Spry2 in HRas-transformed cells decreased Rac1 activation, but independent expression of Spry2 in the non-transformed parental cells had no effect on Rac1, suggesting a specific involvement in the activation of Rac1 by Ras. Silencing Spry2 in HRas(V12)-transformed cells resulted in diminished interaction between HRas and Tiam1, a Rac1-specific nucleotide exchange factor. Expression of constitutively active Rac1 in cells with silenced Spry2 partly reversed the effect of Spry2 down-regulation. Furthermore, loss of Spry2 expression in HRas(V12)-transformed cells augmented the cytotoxicity of the DNA-damaging, chemotherapeutic agent cisplatin, a process that was also reversed by active Rac1. Together, these data show that Spry2 inhibits apoptosis in response to DNA damage by regulating Akt, HDM2, and p53, by a process mediated partly by Rac1.

The biphasic role of the hypoxia-inducible factor prolyl-4-hydroxylase, PHD2, in modulating tumor-forming potential.

Hypoxia is a common feature of solid tumors. The cellular response to hypoxic stress is controlled by a family of prolyl hydroxylases (PHD) and the transcription factor hypoxia-inducible factor 1 (HIF1). To investigate the relationship between PHD and HIF1 activity and cellular transformation, we characterized the expression levels of PHD isoforms across a lineage of cell strains with varying transformed characteristics. We found that PHD2 is the primary functional isoform in these cells and its levels are inversely correlated to tumor-forming potential. When PHD2 levels were altered with RNA interference in nontumorigenic fibroblasts, we found that small decreases can lead to malignant transformation, whereas severe decreases do not. Consistent with these results, direct inhibition of PHD2 was also shown to influence tumor-forming potential. Furthermore, we found that overexpression of PHD2 in malignant fibroblasts leads to loss of the tumorigenic phenotype. These changes correlated with HIF1alpha activity, glycolytic rates, vascular endothelial growth factor expression, and the ability to grow under hypoxic stress. These findings support a biphasic model for the relationship between PHD2 activity and malignant transformation.

hRev7, putative subunit of hPolzeta, plays a critical role in survival, induction of mutations, and progression through S-phase, of UV((254nm))-irradiated human fibroblasts.

Translesion synthesis (TLS) refers to mechanisms by which specialized DNA polymerases incorporate nucleotides opposite fork-blocking lesions and extend replication until standard replicative polymerases take over. The first eukaryotic TLS polymerase discovered, S. cerevisiae Polzeta, consists of catalytic subunit Rev3 and non-catalytic subunit Rev7. Human homologs of these two proteins have been identified. Studies by Lawrence, Maher, and colleagues comparing UV((254nm))-irradiated human fibroblast cell strains expressing high levels of hRev3 antisense to their normal parental strains demonstrated that there was no difference in cell survival, but that the frequency of UV-induced mutations in the derivative strains was 10-fold lower than that of the parental strains, indicating that hRev3 plays a critical role in such mutagenesis. To examine the role of hRev7 in TLS, we generated human fibroblasts expressing hRev7 siRNA, identified two derivative cell strains with significantly reduced levels of hRev7, and compared them to their parental strain and a vector control for cell survival, induction of mutations, and ability to traverse the cell cycle following exposure to UV radiation. Cells with reduced hRev7 were approximately 2-times more sensitive to UV-induced cytotoxicity than the controls, indicating that unlike hRev3, hRev7 plays a protective role for cells exposed to UV radiation. When these cell strains were assayed for the frequency of mutations induced by UV in their HPRT gene, cell stains with reduced hRev7 were 5-times less sensitive to UV-induced mutagenesis than control strains. In addition, when these four strains were synchronized at the G1/S border, released from the block, UV-irradiated, and allowed to traverse the cell cycle, the rate of progression through S-phase of the cell strains with reduced hRev7 was significantly slower than that of the control strains. These data strongly support the hypothesis that hRev7 is required for TLS past UV-photoproducts, and together with hRev3, comprise hPolzeta.

Evidence that sprouty 2 is necessary for sarcoma formation by H-Ras oncogene-transformed human fibroblasts.

Sprouty 2 (Spry2) acts as an inhibitor of receptor tyrosine kinase signaling in various cellular contexts. Interestingly, Spry2 also prevents the c-Cbl-induced degradation of epidermal growth factor receptor (EGFR). We compared human fibroblasts malignantly transformed by overexpression of H-Ras(V12) oncogene to their nontransformed parental cells and found that the malignant cells express a high level of Spry2. These cells also exhibited an increase in the level of EGFR compared with their precursor cells. We found that intact EGFR was required if H-Ras-transformed cells were to grow in the absence of exogenous growth factors or form large colonies in agarose. When we decreased expression of Spry2, using a Spry2-specific shRNA, the H-Ras(V12)-transformed fibroblasts could no longer form large colonies in agarose, grow in reduced levels of serum, or form tumors in athymic mice. The level of active H-Ras in these cells remained unaltered. A similar, but less pronounced, effect in tumor formation was observed when Spry2 was down-regulated in human patient-derived fibrosarcoma cell lines. In H-Ras-transformed cells Spry2 sustained the level and the downstream signaling activity of EGFR. In the parental, non-H-Ras-transformed fibroblasts, expression of Spry2 resulted in the inhibition of H-Ras and ERK activation, suggesting that the positive effect of Spry2 in tumor formation is specific to H-Ras transformation. Co-immunoprecipitation studies with H-Ras-transformed cells revealed that Spry2 and H-Ras interact and that H-Ras interacts with Spry2-binding partners, c-Cbl and CIN85, in a Spry2-dependent manner. These data show that Spry2 plays a critical role in the ability of H-Ras-transformed cells to form tumors in athymic mice.

Evidence that in xeroderma pigmentosum variant cells, which lack DNA polymerase eta, DNA polymerase iota causes the very high frequency and unique spectrum of UV-induced mutations.

Xeroderma pigmentosum variant (XPV) patients have normal DNA excision repair, yet are predisposed to develop sunlight-induced cancer. They exhibit a 25-fold higher than normal frequency of UV-induced mutations and very unusual kinds (spectrum), mainly transversions. The primary defect in XPV cells is the lack of functional DNA polymerase (Pol) eta, the translesion synthesis DNA polymerase that readily inserts adenine nucleotides opposite photoproducts involving thymine. The high frequency and striking difference in kinds of UV-induced mutations in XPV cells strongly suggest that, in the absence of Pol eta, an abnormally error-prone polymerase substitutes. In vitro replication studies of Pol iota show that it replicates past 5'T-T3' and 5'T-U3' cyclobutane pyrimidine dimers, incorporating G or T nucleotides opposite the 3' nucleotide. To test the hypothesis that Pol iota causes the high frequency and abnormal spectrum of UV-induced mutations in XPV cells, we identified an unlimited lifespan XPV cell line expressing two forms of Pol iota, whose frequency of UV-induced mutations is twice that of XPV cells expressing one form. We eliminated expression of one form and compared the parental cells and derivatives for the frequency and kinds of UV-induced mutations. All exhibited similar sensitivity to the cytotoxicity of UV((254 nm)), and the kinds of mutations induced were identical, but the frequency of mutations induced in the derivatives was reduced to

Identification of the promoter of human transcription factor Sp3 and evidence of the role of factors Sp1 and Sp3 in the expression of Sp3 protein.

In a study of the role of transcription factor Sp1 in the formation of tumors by human fibrosarcoma cell lines that overexpress it [Cancer Res., 65 (2005) 1007], we found that expression of an Sp1-specific ribozyme, not only reduced the level of Sp1 protein, but also that of Sp3 protein, and that when the protein levels of these two transcription factors in the fibrosarcoma cell lines were reduced to near that found in normal human fibroblasts, the cell lines could no longer form tumors. An Sp1-specific ribozyme could reduce the level of expression of both Sp1 protein and Sp3 protein if the promoter of the Sp1 gene and that of the Sp3 gene both have Sp1/Sp3 transcription factor binding sites and if such sites are critically responsible for the level of expression of both Sp1 and Sp3 protein in the cells. The Sp1 minimal promoter has been identified and it has two Sp1/Sp3 sites [J. Biol. Chem. 276 (2001) 22126]. To characterize the Sp3 promoter, we isolated 2.1 kb of the 5'-flanking region of the Sp3 gene, which contains Sp1/Sp3 binding sites, and using an expression reporter assay, showed that it has promoter activity. We then systematically reduced the size of the 5' flanking region, and determined that the nt-339 to nt-39 fragment, which contains an Sp1/Sp3 binding site at nt-181 and another at nt-168, retained the same promoter activity as the 2.1 kb region. Electrophoretic mobility shift assays indicated that both Sp3 protein and Sp1protein bind to these two sites. By mutating either or both of these binding sites, we showed using the reporter assay that each site is required for full promoter activity. We then designed an Sp3-specific ribozyme, expressed it in a human fibrosarcoma cell line in which Sp1 protein and Sp3 protein are expressed at high levels, and found that, indeed, the level of expression of both proteins was significantly reduced.

Down-regulation of overexpressed sp1 protein in human fibrosarcoma cell lines inhibits tumor formation.

Sp1 is a transcription factor for many genes, including genes involved in tumorigenesis. We found that human fibroblast cells malignantly transformed in culture by a carcinogen or by stable transfection of an oncogene express Sp1 at 8-fold to 18-fold higher levels than their parental cells. These cell lines form fibrosarcomas in athymic mice with a very short latency, and the cells from the tumors express the same high levels of Sp1. Similar high levels of Sp1 were found in the patient-derived fibrosarcoma cell lines tested, and in the tumors formed in athymic mice by these cell lines. To investigate the role of overexpression of Sp1 in malignant transformation of human fibroblasts, we transfected an Sp1 U1snRNA/Ribozyme into two human cell lines, malignantly transformed in culture by a carcinogen or overexpression of an oncogene, and into a patient-derived fibrosarcoma cell line. The level of expression of Sp1 in these transfected cell lines was reduced to near normal. The cells regained the spindle-shaped morphology and exhibited increased apoptosis and decreased expression of several genes linked to cancer, i.e., epithelial growth factor receptor, urokinase plasminogen activator, urokinase plasminogen activator receptor, and vascular endothelial growth factor. When injected into athymic mice, these cell lines with near normal levels of Sp1 failed to form tumors or did so only at a greatly reduced frequency and with a much longer latency. These data indicate that overexpression of Sp1 plays a causal role in malignant transformation of human fibroblasts and suggest that for cancers in which it is overexpressed, Sp1 constitutes a target for therapy.

Sp1 regulates expression of MET, and ribozyme-induced down-regulation of MET in fibrosarcoma-derived human cells reduces or eliminates their tumorigenicity.

Changes in expression of hepatocyte growth factor (HGF) and its receptor, MET, are associated with formation and malignant progression of human tumors. In the present study, 10 of 11 human fibrosarcoma cell lines tested expressed significantly higher levels of MET than were found in a series of normal human fibroblast lines. Still more significant, MET was constitutively phosphorylated in all 11 fibrosarcoma lines, whereas the normal fibroblasts exhibited very low levels of the phosphorylated form. All the cell lines expressed HGF mRNA. To determine the role of MET and/or HGF in tumorigenesis, a fibrosarcoma line expressing high levels of MET protein and low levels of HGF/NK2 mRNA was stably transfected with a hammerhead ribozyme targeting MET. In addition, a fibrosarcoma line expressing high levels of both MET protein and HGF/NK2 mRNA was transfected with a ribozyme targeting MET, or with a ribozyme targeting MET and another targeting HGF. The transfectant cell lines no longer formed tumors, or did so at a greatly reduced frequency and/or longer latency. Because Sp1 is a transcription factor for MET, we assayed the cell lines for their level of Sp1 protein. Sp1 was markedly overexpressed in 7 of the 11 fibrosarcoma lines compared to normal fibroblast lines. Deletion analysis and site-directed mutagenesis of the MET promoter revealed that tandem Sp1 sites in the proximal promoter are critical for transcription of MET. Increased expression of Sp1 in a normal human fibroblast line containing a MET promoter-luciferase construct resulted in a dose-dependent increase in luciferase. Conversely, inhibition of Sp1 binding to DNA in a fibrosarcoma cell line, using an Sp1 decoy, dramatically reduced MET expression. Taken together, these results indicate that in human fibrosarcoma cells, high levels of the phosphorylated form of MET are required for tumor formation and that Sp1 can function to control the level of MET.