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Gap junction proteins (connexins) are expressed in a cell-specific manner and expression is often reduced in neoplastic cells. We investigated the mechanisms of connexin32 (Cx32) and connexin43 (Cx43) expression in hepatic cells using MH1C1 rat hepatoma cells and freshly isolated, adult rat hepatocytes that express Cx32 but not Cx43 and WB-F344 rat liver epithelial cells that express Cx43 but not Cx32. Southern blotting after DNA restriction with MspI and HpaII indicated that two MspI/HpaII restriction sites in the Cx32 promoter (positions -147 and -847) were methylated in WB-F344 cells, but not in MH1C1 cells or hepatocytes. In contrast, an MspI/HpaII restriction site in the Cx43 promoter (position -38) was methylated in MH1C1 cells, but not in WB-F344 cells or hepatocytes. Transient transfection of the cell lines with connexin promoter-luciferase constructs indicated that the Cx32 promoter was 7-fold more active in MH1C1 cells and the Cx43 promoter was 5-fold more active in WB-F344 cells. These results suggest that transcription of Cx32 and Cx43 in hepatic cells is controlled by promoter methylation and by cell-specific transcription factors. Similar mechanisms may be involved in the reduced expression of these genes frequently observed in neoplastic cells. [ABSTRACT FROM PUBLISHER]

The mechanisms that underly the regulation of human CYP1A1 have merited considerable attention because of their association both with toxic outcomes and the etiology of several cancers. Previous work conducted in this laboratory has identified a negative regulatory element (NRE) in the 5' region of this gene that appeared to modulate CYP1A1 transcriptional activity. This NRE is present in two functional copies, a high affinity 21-bp palindrome centred at position -784, and an additional element found within a GC-rich region between position -728 and -558. In this report, the regulatory function of the NREs in the context of the CYP1A1 promoter was evaluated. This was accomplished by substituting mutated elements for the corresponding wild-type element in a vector that contained human CYP1A1 sequences positions -1140 to +59 directing the transcription of the chloramphenicol acetyltransferase reporter gene. Expression vectors containing specific mutations in each or both NREs were characterized. We show that eliminating the binding of the CYP1A1 repressor protein to one or both repressor motifs results in a significant 2- to 3-fold increase in the inducibility over that observed with only one site mutated, the effect was not additive. Such aberrant transcriptional activity correlates with the highly inducible aryl hydrocarbonhydroxylase phenotype that is a reported marker for individuals predisposed to lung cancer. Mutation of the NRE, or more likely, the cognate repressor protein(s), may provide a genetic basis for this phenotype. [ABSTRACT FROM PUBLISHER]

Background: Adjuvant trastuzumab (Herceptin) treatment of breast cancer patients significantly improves their clinical outcome. Vaccination is an attractive alternative approach to provide HER-2/neu (Her2)-specific antibodies and may in addition concomitantly stimulate Her2-reactive T-cells. Here we report the first administration of a Her2-plasmid DNA (pDNA) vaccine in humans.Patients and Methods: The vaccine, encoding a full-length signaling-deficient version of the oncogene Her2, was administered together with low doses of GM-CSF and IL-2 to patients with metastatic Her2-expressing breast carcinoma who were also treated with trastuzumab. Six of eight enrolled patients completed all three vaccine cycles. In the remaining two patients treatment was discontinued after one vaccine cycle due to rapid tumor progression or disease-related complications. The primary objective was the evaluation of safety and tolerability of the vaccine regimen. As a secondary objective, treatment-induced Her2-specific immunity was monitored by measuring antibody production as well as T-cell proliferation and cytokine production in response to Her2-derived antigens.Results: No clinical manifestations of acute toxicity, autoimmunity or cardiotoxicity were observed after administration of Her2-pDNA in combination with GM-CSF, IL-2 and trastuzumab. No specific T-cell proliferation following in vitro stimulation of freshly isolated PBMC with recombinant human Her2 protein was induced by the vaccination. Immediately after all three cycles of vaccination no or even decreased CD4+ T-cell responses towards Her2-derived peptide epitopes were observed, but a significant increase of MHC class II restricted T-cell responses to Her2 was detected at long term follow-up. Since concurrent trastuzumab therapy was permitted, lambda-subclass specific ELISAs were performed to specifically measure endogenous antibody production without interference by trastuzumab. Her2-pDNA vaccination induced and boosted Her2-specific antibodies that could be detected for several years after the last vaccine administration in a subgroup of patients.Conclusion: This pilot clinical trial demonstrates that Her2-pDNA vaccination in conjunction with GM-CSF and IL-2 administration is safe, well tolerated and can induce long-lasting cellular and humoral immune responses against Her2 in patients with advanced breast cancer. [ABSTRACT FROM AUTHOR]

Background: To gain a better understanding of the effects of therapeutic agents on the tumor microenvironment in invasive cancers, we developed a co-culture model from an invasive lobular carcinoma. Tumor cells expressing HER2/neu organize in nests surrounded by alpha-Smooth Muscle Actin (alpha-SMA) expressing tumor stroma to resemble the morphology of an invading tumor. This co-culture, Mammary Adenocarcinoma Model (MAM-1) maintains a 1:1 ratio of HER2/neu positive tumor cells to alpha-SMA-reactive stromal cells and renews this configuration for over 20 passages in vitro.Methods: We characterized the cellular elements of the MAM-1 model by microarray analysis, and immunocytochemistry. We developed flow cytometric assays to evaluate the relative responses of the tumor and stroma to the tyrosine kinase inhibitor, Iressa.Results: The MAM-1 gene expression profile contains clusters that represent the ErbB-2 breast cancer signature and stroma-specific clusters associated with invasive breast cancers. The stability of this model and the ability to antigenically label the tumor and stromal fractions allowed us to determine the specificity of Iressa, a receptor tyrosine kinase inhibitor, for targeting the tumor cell population. Treatment resulted in a selective dose-dependent reduction in phospho-pMEK1/2 and pp44/42MAPK in tumor cells. Within 24 h the tumor cell fraction was reduced 1.9-fold while the stromal cell fraction increased >3-fold, consistent with specific reductions in phospho-pp44/42 MAPK, MEK1/2 and PCNA in tumor cells and reciprocal increases in the stromal cells. Erosion of the tumor cell nests and augmented growth of the stromal cells resembled a fibrotic response.Conclusion: This model demonstrates the specificity of Iressa for HER2/neu expressing tumor cells versus the tumor associated myofibroblasts and is appropriate for delineating effects of therapy on signal transduction in the breast tumor microenvironment and improving strategies that can dually or differentially target the tumor and stromal elements in the microenvironment. [ABSTRACT FROM AUTHOR]

In situ expression of a foreign antigen and an immune-modulating cytokine by intratumoral DNA electroporation was tested as a cancer therapy regimen. Transgene expression in the tumors was sustained for 2-3 weeks after intratumoral electroporation with mammalian expression plasmid containing firefly luciferase cDNA. Electroporation with cDNA encoding tetanus toxin fragment C (TetC) induced tetanus toxin-binding antibody, demonstrating immune recognition of the transgene product. Intratumoral electroporation with TetC and IL-12 cDNA after mice were treated with CD25 mAb to remove regulatory T cells induced IFN-gamma producing T-cell response to tumor-associated antigen, heavy inflammatory infiltration, regression of established tumors and immune memory to protect mice from repeated tumor challenge. Intratumoral expression of immune-modulating molecules may be most suitable in the neoadjuvant setting to enhance the therapeutic efficacy and provide long-term protection.

Immune tolerance to tumor-associated self-antigens poses a major challenge in the ability to mount an effective cancer vaccine response. To overcome immune tolerance to HER-2, we formulated DNA vaccines that express both human HER-2 and heterologous rat Neu sequences in separate plasmids or as single hybrid constructs that encode HER-2/Neu fusion proteins. Candidate vaccines were tested in Her-2 transgenic (Tg) mice of BALB/c (BALB), BALB/cxC57BL/6 F1 (F1), or C57BL/6 (B6) background, which exhibit decreasing immune responsiveness to HER-2. Analysis of various cocktails or hybrid vaccines defined a requirement for particular combination of HER/2/Neu sequences to effectively prime immune effector cells in HER-2 Tg mice. In B6 HER-2 Tg mice, rejection of HER-2-positive tumors protected mice from HER-2-negative tumors, providing evidence of epitope spreading. Our findings show that a strategy of combining heterologous antigen with self-antigens could produce a potent DNA vaccine that may be applicable to other tumor-associated antigens.

Her-2/neu(+) tumor cells refractory to antibody or receptor tyrosine kinase inhibitors are emerging in treated patients. To investigate if drug resistant tumors can be controlled by active vaccination, gefitinib and antibody sensitivity of four neu(+) BALB/c mouse mammary tumor lines were compared. Significant differences in cell proliferation and Akt phosphorylation were observed. Treatment-induced drug resistance was associated with increased chromosomal aberrations as shown by spectral karyotyping analysis, suggesting changes beyond neu signaling pathways. When mice were immunized with pneuTM encoding the extracellular and transmembrane domains of neu, antibody and T-cell responses were induced, and both drug-sensitive and drug-resistant tumor cells were rejected. In T-cell-depleted mice, drug-sensitive tumors were still rejected by vaccination, but drug-refractory tumors survived in some mice, indicating their resistance to anti-neu antibodies. To further test if T cells alone can mediate tumor rejection, mice were immunized with pcytneu encoding full-length cytoplasmic neu that is rapidly degraded by the proteasome to activate CD8 T cells without inducing antibody response. All test tumors were rejected in pcytneu-immunized mice, regardless of their sensitivity to gefitinib or antibody. Therefore, cytotoxic T lymphocytes activated by the complete repertoire of neu epitopes were effective against all test tumors. These results warrant Her-2 vaccination whether tumor cells are sensitive or resistant to Her-2-targeted drugs or antibody therapy.

The HER2/neu oncogene is an important diagnostic and prognostic factor and therapeutic target in breast and other cancers. We developed and characterized a breast cancer cell line (Bam1a) that overexpresses the activated HER2/neu and ErbB-3 and has a gene expression profile consistent with the ErbB-2 genetic signature. We evaluated the effects of the epidermal growth factor receptor (EGFR)/HER2 inhibitor, gefitinib, on this breast tumor line in vitro and in vivo. We characterized the effects of gefitinib on EGFR, HER2, and ErbB-3 phosphorylation by Western blot and determined the effects on downstream signaling through growth, survival, and stress pathways and the effect on proliferation, cell cycle, and apoptosis. Gefitinib treatment diminished phosphorylation of the ErbB-3 > EGFR > HER2/neu and signal transducers and activators of transcriptions in a dose-dependent fashion. Downstream mitogenic signaling through mitogen-activated protein (MAP)/extracellular signal regulated kinase kinase, p44/42 MAP kinase (MAPK) and stress signaling through c-Jun-NH(2)-kinase (JNK) 1 and c-Jun was impaired (1 micromol/L, 4-24 h), leading to cytostasis and cell cycle arrest within 24 h by decreased cyclin D1, cyclin B1, and p(Ser795)Rb and increased p27. Proliferation and colony formation were inhibited at 0.5 and 1 micromol/L, respectively, and correlated with altered gene expression profiles. Diminished survival signaling through Akt, induction of bim, loss of connexin43, and decreased production of vascular endothelial growth factor-D preceded caspase-3 and poly(ADP)ribose polymerase (PARP) cleavage and apoptosis (>50% 2 micromol/L, 48 h). Oral administration of gefitinib was able to prevent the outgrowth of Bam1a tumor cells from palpable lesions, shrink established tumors, eliminate HER2 and HER3 phosphorylation, and decrease MAPK and Akt signaling in vivo. A variant of the Bam1a cell line, IR-5, with acquired ability to grow in 5 micromol/L gefitinib was developed and characterized. IR-5 bears a novel point mutation in the HER2/neu that corresponds to a L726I in the ATP-binding pocket and correlates with a log decrease in sensitivity to gefitinib, increased heterodimerization with EGFR and HER3, and impaired down-regulation. Gene expression profiling of IR-5 showed increased expression of EMP-1, NOTCH-1, FLT-1, PDGFB, and several other genes that may contribute to the resistant phenotype and sustain signaling through MAPK and Akt. This model will be useful in understanding the differences between intrinsic drug sensitivity and acquired resistance in the context of therapeutic strategies that target oncogene addicted diseases.

Lymphocytes polarize for motility by developing a broad anterior, where lamellipodia arise, and a simple stalk-like posterior appendage, the uropod. Through time-lapse analysis of normal and leukemic human T cells, it was found that this polarized form is maintained by a mechanism that excludes lamellipodia from the uropod. Lamellipodia regularly traveled rearward to encroach upon the uropod but disassembled abruptly at the uropod border. This exclusion of lamellipodia from the uropod required the Rho-family guanosine triphosphatase Cdc42. Reduction of Cdc42 activity by expression of dominant-negative Cdc42 resulted in "two headed" cells in which lamellipodia persisted at the distal end of the uropod. Random and chemotactic motility were impaired. Increased Cdc42 activity, induced by expression of activated, mutant Cdc42, was accompanied by a general loss of lamellipodia. The results suggest that one role of Cdc42 in lymphocyte motility is to preserve polarity by concentrating lamellipodial disassembly signals in the uropod.

Purpose: The purpose is to identify gene expression patterns induced by docetaxelin head and neck squamous carcinoma (HNSCC) cells using high throughput techniques., Experimental Design: HNSCC cells were treated with docetaxel or solvent. After mRNA extraction, cDNA fluorescent (Cy3 or Cy5)-labeled probes were synthesized. Then, Cy3 and Cy5-labeled samples were hybridized onto a microarray slide. The fluorescent images were scanned and analyzed for quantification. PowerBlot immunoblotting technique was used to measure protein expression level. Using this dual approach, we focused on genes in established pathways (cell cycle, apoptosis, angiogenesis, and signal transduction) of tumorigenesis and confirmed these results with conventional techniques., Results: Using cDNA microarray, we found that docetaxel altered the expression of >100 genes in HNSCC cells. A total of 153 of 1191 genes was found to have altered expression in either HN12 (n = 102), HN30 (n = 72), or both (n = 21) by docetaxel. For the PowerBlot analysis, a subset of genes (n = 46) in the cDNA microarray analysis and an additional 98 genes in the cell cycle, apoptosis, angiogenesis, and signal transduction pathways were chosen. We found that PowerBlot data agreed with cDNA microarray in 65% of genes examined. The expression of a cell cycle inhibitor (p19) and promoters (cyclin A, cyclin B1, and cyclin E2F) were increased and decreased, respectively. Apoptosis induced by docetaxel was independent of p53 and, in part, related to increased Fas expression. Both vascular endothelial growth factor secretion and basic fibroblast growth factor expression were inhibited by docetaxel, whereas thrombospondin-1 expression was increased by docetaxel. Epidermal growth factor receptor, activated epidermal growth factor receptor, and activated c-Jun NH(2)-terminal kinase expression was lowered by docetaxel. Activated extracellular signal-regulated kinase was elevated by docetaxel, but not total extracellular signal-regulated kinase levels., Conclusions: The identification of altered gene expression induced by docetaxel demonstrates additional biological activity in HNSCC cells, and the altered expression of these genes may serve as potential biomarkers to both predict clinical activity and provide information regarding potential efficacy of adding novel agents.

Purpose: The purpose of this study was to evaluate the biological mechanisms of docetaxel (TXT) on salivary gland carcinoma., Experimental Design: The effects of TXT on a spontaneous murine salivary carcinoma were determined. Proliferation, cell cycle regulation, connexin43 expression, gap-junctional intercellular communication, apoptosis, and Fas receptor (FasR) expression were measured., Results: We characterized a spontaneous mouse salivary gland carcinoma (SGC1). SGC1 is a poorly differentiated carcinoma that originated from the parotid gland of a BALB/c mouse. SGC1 cells were cultured and found to be immortal past 30 passages. Initially, cells formed tumor nodules in severe combined immunodeficient (SCID) mice. Afterward, SGC1 cells that were subcultured from SCID tumors readily formed colonies in soft agar and were highly tumorigenic in SCID mice and immune-competent BALB/c hosts. Dose response for TXT with respect to growth suppression, G(2)-M cell cycle arrest, and apoptosis was found. Induction of apoptosis by TXT coincided with an increase in cell surface FasR expression. Up-regulation of FasR with lower doses of TXT rendered cells susceptible to FasR agonist antibody-mediated apoptosis. In the absence of TXT, anti-FasR antibodies were completely without effect, suggesting that TXT is critical for priming apoptosis mediated through the Fas pathway. In addition, gap-junctional intercellular communication was augmented by TXT in SGC1 cells concomitant with increased connexin43 expression and membrane localization., Conclusions: We have identified several novel targets of TXT that contribute to its antitumor activity in poorly differentiated salivary gland carcinoma. These results suggest that TXT may be appropriate for additional in vivo studies and clinical trials in patients with salivary cancers.

Gap junctional intercellular communication (GJIC) and the expression of gap junction proteins (connexins) are frequently decreased in neoplastic cells and have been increased by cAMP and retinoids. GJIC and connexin expression were investigated in early passage normal human ovarian surface epithelial (HOSE) cells, human ovarian adenocarcinoma cell lines (CaOV-3, NIH:OVCAR-3, SK-OV-3 and SW626) and surgical specimens of human serous cystadenocarcinomas. We hypothesized that GJIC and connexin expression would be decreased in neoplastic cells and would be increased by cAMP and retinoic acid. Cultured HOSE cells exhibited extensive fluorescent dye-coupling and connexin43 (Cx43) expression; other connexins were not detected. The ovarian adenocarcinoma cell lines had little dye-coupling or connexin expression. Deletions and rearrangements of the Cx43 gene were not detected by Southern blotting in the carcinoma lines. N6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate and all-trans-retinoic acid inhibited cell proliferation, but did not enhance GJIC or Cx43 expression. Surface epithelial cells of benign ovaries expressed Cx43, but this expression was barely detectable in ovarian serous cystadenocarcinomas. Thus, normal HOSE cells had extensive GJIC and Cx43 expression whereas ovarian carcinoma cells had less and cAMP and retinoic acid did not change these, although both agents inhibited cell growth.

Previous studies in our laboratory identified a negative regulatory domain in the 5'-flanking region of the human CYP1A1 gene containing two negative regulatory elements (NRE). Characterization of one of these elements revealed three nuclear protein binding regions: a 21-bp palindrome with a point of symmetry at -784 and two guanine- and cytosine-rich elements that flank the palindrome. Functional studies suggested the palindrome is critical for transcriptional repression, whereas the guanine- and cytosine-rich sequences play a secondary role. In this study, the interaction between nuclear proteins and the CYP1A1 NRE was further defined. Electrophoretic mobility shift assays (EMSA) indicated that the NRE -784 palindrome alone, but not the guanine- and cytosine-rich sequences minus the palindrome, was capable of specific nuclear protein binding. Competitive cotransfection experiments confirmed this observation in intact cells. Specific residues important for DNA-protein interactions were identified by site-directed mutagenesis and competitive EMSA. The loss of specific protein binding was also correlated with the loss of negative regulatory activity in a transient-expression assay. Finally, competitive EMSA was performed with consensus oligonucleotides for known transcription factors. An NF-Y consensus sequence efficiently competed with the NRE probe for specific nuclear protein binding. EMSA supershift analyses indicate that a protein immunologically related to NF-YB is part of the specific nuclear protein complex binding the human CYP1A1 NRE. These studies have refined our understanding of the sequences critical for the transcriptional repression of human CYP1A1. To our knowledge, this is also the first report implicating a member of the NF-Y transcription factor family in negative gene regulation.

Low-dose gamma radiation stimulates expression of phenotypic characteristics in B16 melanoma cells which regulate metastatic potential. A transient increase in the expression of an integrin receptor (alpha IIb beta 3) was observed after exposure of B16 melanoma cells to 0.25 to 2.0 Gy of gamma radiation. This increased receptor expression resulted in enhanced adhesion of tumor cells to fibronectin in vitro and increased experimentally induced metastasis in vivo. In this report, we determined a role for the 12-lipoxygenase metabolite, 12-HETE, in radiation-enhanced metastasis. A significant increase in biosynthesis of 12-HETE in B16 melanoma cells was detected < 5 min after exposure to 0.5 Gy gamma radiation. We then determined that radiation-enhanced expression of alpha IIb beta 3 integrin and adhesion of B16 melanoma cells to fibronectin in vitro and metastasis in vivo were reduced by treatment of the cells with the lipoxygenase inhibitor NDGA prior to irradiation. These findings suggest that low-dose radiation, at levels comparable to those used in fractionated or hyper-fractionated radiotherapy, increases the metastatic potential of surviving tumor cells via a rapid and transient alteration in lipoxygenase metabolism of arachidonic acid and surface expression of an integrin receptor.

We investigated the effects of nonlethal gamma radiation on the metastatic potential of the murine tumor cell line, B16 melanoma. The ability of B16 cells to adhere to fibronectin, which is in part mediated by the alpha IIb beta 3 integrin receptor, is predictive of metastatic potential. We determined that exposure to 0.25-2.5 Gy gamma radiation significantly enhanced B16 cell adhesion to fibronectin. The radiation-enhanced adhesion was dependent on enhanced expression of the alpha IIb beta 3 integrin. We observed that 15 min after 0.5 Gy radiation, 99% of irradiated B16 tumor cells were positively labeled with monoclonal antibodies directed against alpha IIb beta 3 compared to 22% of sham-irradiated cells. Radiation-enhanced expression of the alpha IIb beta 3 receptor is reversible and down-regulation begins within 2-4 h postirradiation. Finally, we found that irradiation significantly enhanced the ability of B16 cells to form metastases in a lung colony assay. It is concluded that a relationship exists between radiation effects on the B16 tumor cells, alpha IIb beta 3 receptor expression, adhesion in vitro, and metastasis in vivo. We suggest that low-dose radiation, at levels comparable to those used in fractionated or hyperfractionated radiotherapy, may alter the metastatic phenotype and potential of surviving tumor cells via a rapid alteration in their surface expression of alpha IIb beta 3 integrin receptors.