There are many significant differences in the structural and functional anatomy of the nasal cavity of man and laboratory animals. Some of the differences may be responsible for the species-specific nasal lesions that are often observed in response to inhaled toxicants. This paper reviews the comparative anatomy, physiology and pathology of the nasal cavity of the rat, mouse, dog, monkey and man, highlighting factors that may influence the distribution of nasal lesions. Gross anatomical variations such as turbinate structure, folds or grooves on nasal walls, or presence or absence of accessory structures, may influence nasal airflow and species-specific uptake and deposition of inhaled material. In addition, interspecies variations in the morphological and biochemical composition and distribution of the nasal epithelium may affect the local tissue susceptibility and play a role in the development of species-specific nasal lesions. It is concluded that, while the nasal cavity of the monkey might be more similar to that of man, each laboratory animal species provides a model that responds in a characteristic and species-specific manner. Therefore for human risk assessment, careful consideration must be given to the anatomical differences between a given animal model and man.

4116 Background: Virulizin (V) is a novel antitumor immune modulator that improves survival in pancreatic cancer patients (pts) as monotherapy. A double-blind, multicenter, randomized, phase III study was conducted to evaluate the survival benefits and safety of V in combination with gemcitabine (G) in pts with advanced pancreatic cancer. Methods: Chemo-naive pts with locally advanced or metastatic pancreatic cancer with ECOG Performance Status (PS) of 0, 1 or 2 were enrolled. Pts were randomized to receive intramuscular injections of either V or placebo (P) 3 times weekly + G (1,000 mg/m2 weekly ×7 with 1 week rest, then weekly ×3 q4w). Randomization was stratified according to ECOG PS (0 or 1, and 2) and extent of disease (locally advanced and metastatic). Pts who showed no clinical benefit or were intolerant to G entered 2nd-line therapy (stage 3), in which pts continued to receive either V or P alone or with 5-FU, or best supportive care. The primary endpoint was survival, defined as time from baseline/treatment day 1 to time of death from any cause. Results: The intent to treat (ITT) population comprised 434 pts, of which 377 were efficacy evaluable (EE). Median overall survival for V + G was 6.3 months for the ITT population (6.8 months for EE pts) and 6 months for P + G for both ITT and EE pts. While differences in survival times were not statistically significant, exploratory analysis showed encouraging results in specific subgroups treated with V + G ( table ). Importantly, a significant difference was found in stage 3 pts who received V in a salvage setting compared to pts who received P. Conclusions: Pancreatic cancer pts with either low ECOG PS or metastatic cancer showed a survival benefit when treated with V + G, which was significant in pts who continued to receive V as a salvage therapy. Further studies in these targeted patient populations are being considered. [Table: see text] No significant financial relationships to disclose.

One major problem associated with collecting whole blood from patients for use as a source of RNA in gene expression studies is that the RNA degrades during collection and storage. Preservation of RNA quality is vital in such studies because the stability of the RNA ultimately affects analysis of gene expression. In this study the PAXgene (TM) blood collection system was compared with a standard erythrocyte lysis method for isolating RNA from blood samples. The methods were compared in terms of RNA yield, RNA stabilization, and DNA contamination. The study also included the downstream application to RTPCR analysis for relative mRNA expression levels of the ribonucleotide reductase subunits R1 and R2. The results show that blood collection in conventional collection tubes, and leukocyte isolation by erythrocyte lysis lead to significant degradation of RNA. Our findings confirm the ability of PAXgene (TM) to stabilize RNA in whole blood; however, RNA extracted by the PAXgene (TM) method contained significant DNA contamination. Given the low basal expression of the target genes analyzed in this study,, contaminating DNA could potentially affect accurate interpretation of RT-PCR data. As a result, the PAXgene (TM) protocol was optimized to include off-column DNase treatments, which yielded high-quality RNA suitable for gene expression studies. Furthermore, the results suggest that RNA isolation with PAXgene (TM) is advantageous compared to traditional extraction methods for RT-PCR analysis of large or different-sized amplicons.

4257 Purpose: V is stimulates macrophages and monocytes in vitro and in vivo. Pancreatic cancer patients treated with V in phase I/II studies showed increased 6 and 12 months survival vs historical controls. V administration was associated with increased NK cell activity in patients showing the best response to V. Hence, NK cell function may be a mediator of V anti-tumor activity. Methods: To determine if V induces NK cell infiltration into tumors, CD-1 nude mice with Capan-1 pancreatic tumor xenografts were treated with V; tumor samples were analyzed by flow cytometry using NK cell-specific antibodies. Anti-tumor activity and NK cell infiltration were also evaluated in human tumor xenografts in CD-1 nude mice depleted of macrophages. Immunohistochemical staining of tumor xenografts was performed to further assess NK cell and macrophage activity. Results: V increased NK cell infiltration of tumors (59.4 – 116.0%). Macrophage depletion reduced both NK cell infiltration of tumors, and anti-tumor activity of V (P=0.1632). V-treated mice showed a significant increase in infiltration of F4/80+ (macrophages) and NK1.1+ (NK) cells, vs saline treated controls. Increased NK1.1+ cell infiltration occurred early in V treatment, and correlated with an early marker for tumor apoptosis. V treatment also resulted in a significant increase in the NK cell number in the spleen. Conclusion: Macrophage-mediated NK cell activation/recruitment is implicated in the mechanism of action of V.. These results suggest that V mediates a sustained infiltration of NK cells and macrophages into tumors. Low NK cell activity may be a risk factor for malignancy and metastases, and a negative prognostic indicator in pancreatic cancer. NK cell function and other immune activities are being assessed as biologic markers of clinical response in a Phase III trial in pancreatic cancer as a first line combination therapy with gemcitabine. NK cell function will be correlated with clinical response parameters. No significant financial relationships to disclose.

Although clotrimazole (CLT), an antifungal drug, inhibits tumor cell proliferation and angiogenesis, its clinical application is hampered by significant hepatotoxicity due to the presence of an imidazole moiety. In our attempts to develop CLT analogs that are devoid of imidazole and are as efficacious as CLT, one pharmacophore designated NC381 was generated and shown to inhibit tumor cell growth via a mechanism similar to that of CLT. In vitro, treatment of NCI-H460 nonsmall cell lung cancer (NSCLC) cells with NC381 inhibited growth in a time-dependent manner. Flow cytometric analysis demonstrated that the decrease in cell growth was associated with inhibition of cell cycle progression at the G(1)-S phase transition, resulting in G(0)-G(1) arrest. There was a concomitant inhibition of cyclin D1 expression and subsequent reduction in the formation of the cyclin D1-CDK4 complex. Consistent with a decrease in the cyclin D1-CDK4 complex, NC381 treatment resulted in significant inhibition of pRb phosphorylation. There also were changes in the activity of cell cycle-related proteins, including p16(Ink4) and p27(Kip1). Together, these results are consistent with a model in which NC381 arrests cell cycle progression via inhibition of the pathway that promotes exit from the G(1) phase of the cell cycle. Furthermore, the clinical applicability of NC381 was evaluated in an in vivo murine xenograft model of human NSCLC (NCI-H460). NC381 treatment resulted in significant inhibition of tumor growth. Given the poor prognosis and the limited treatment options available, the present results underscore the potential of NC381 in the treatment of human NSCLC.

Ribonucleotide reductase is the enzyme responsible for the reduction of ribonucleotides to their corresponding deoxyribonucleotides for DNA synthesis. Ribonucleotide reductase is a multisubunit complex containing two polypeptides, R1 and R2. In addition to catalytic and allosteric regulatory functions, the R1 subunit appears to act as a novel tumor suppressor. Previous studies demonstrated that overexpression of mouse R1 resulted in suppression of tumorigenicity and metastatic potential, whereas expression of antisense RNA, complementary to R1 mRNA, increased anchorage-independent growth of ras-transformed NIH 3T3 cells. The current study investigated the potential of R1 gene therapy for human cancer using a recombinant adenovirus encoding the human R1 gene (rAd5-R1). Recombinant viruses were constructed by FLP-mediated site-specific recombination and demonstrated high infectivity of a human colon carcinoma cell line (Colo320 HRS), as assessed by expression of a viral encoded beta-Gal gene (rAd5-LacZ). R1mRNA and protein were overexpressed in Colo320 HRS cells infected with rAd5-R1 compared with untreated or rAd5-LacZ-infected cells. Infection with rAd5-R1 inhibited Colo320 HRS cell proliferation, in vitro, in a time- and dose-dependent manner. When Colo320 HRS cells were treated with rAd5-R1, before injection into CD-1 mice, there was complete inhibition of tumor growth compared with treatment with rAd5-LacZ. Furthermore, intratumoral injection of rAd5-R1 into Colo320 HRS tumor xenografts inhibited tumor growth in CD-1 mice compared with rAd5-LacZ treated mice (P = 0.0001). These results demonstrate gene-specific antitumor effects of R1 and suggest that rAd5-R1 gene therapy has the potential to improve currently available treatments for colon cancer.

Virulizin is a novel biological response modifier (BRM) approved for the treatment of melanoma and is currently in a phase III clinical trial against advanced pancreatic cancer. The purpose of this study was to define the anti-cancer activity of Virulizin against a number of solid human tumors. The therapeutic effect of Virulizin was evaluated in mouse xenograft models, and the results demonstrate that Virulizin has high efficacy against breast, ovarian and prostate tumor xenografts. Seventy-seven percent inhibition, with an optimal T/C value of 24.8%, was observed in human beast MDA-MB-231 xenografts; treated with Virulizin as compared to saline-treated controls (p = 0.0004). In human ovarian SK-OV-3 tumor xenografts, administration of Virulizin inhibited tumor growth by 77.6% compared to saline controls (p = 0.0439). Furthermore, high anti-tumor activity was also demonstrated in DU145 and PC-3 prostate tumor xenografts, as indicated by 72.6 and 49.1% suppression of tumor growth (versus saline controls, p = 0.0007 or p = 0.0049), respectively. Direct comparisons with the anti-tumor activities of conventional drugs demonstrated that Virulizin has higher or equal efficacy against all four tumors tested. Finally, addition of Virulizin into co-cultures of tumor cells and macrophages stimulated the cytolytic activity of the macrophages against the tumor cells in a dose-dependent manner. This result suggests that stimulation of immune cells is at least part of the anti-tumor mechanism of action of Virulizin. These results clearly demonstrate that Virulizin inhibits the growth of human breast, ovarian and prostate tumors, indicating great potential for expansion of the clinical indications for this novel BRM.

Virulizin, a novel biological response modifier (BRM), has been demonstrated to have a high level of anti-tumor activity against pancreatic cancer and melanoma in many clinical trials and preclinical studies. However, its anti-tumor mechanism has not been fully elucidated. The purpose of this study was to define the mechanism of Virulizin anti-tumor activity in cultures and in a murine xenograft model. The presence of Virulizin stimulated in a dose-dependent manner the cytolytic activity against tumor cells by splenocytes and macrophages, but not by non-adherent splenocytes. The cytotoxic activity of macrophages was significantly increased (approximately 5-fold) in cultures containing 2.5% of Virulizin compared to that of cultures without Virulizin (p<0.001). An increase of 21% in the protease secretion was observed in Virulizin (2.5%)-stimulated macrophages compared to PBS-treated cells (p<0.0001). Moreover, the anti-tumor efficacy of Virulizin observed in CD-1 nude mice was abrogated in mice that were depleted of macrophages, thus stimulation of macrophages may be one mechanism through which Virulizin acts. These results suggest that macrophages may play a critical role in the anti-tumor activity of Virulizin.

The mechanisms through which the oncoprotein c-Myc initiates locus-specific gene amplification are not understood. When analysing the initiation mechanism of c-Myc-dependent amplification of the mouse ribonucleotide reductase R2 (R2) gene, we observe c-Myc-dependent initiation of illegitimate DNA replication of the R2 gene. We demonstrate multiple simultaneous c-Myc-induced R2 replication forks, whereas R2 normally replicates with a single fork. In contrast, cyclin C replicates with only a single replication fork irrespective of c-Myc deregulation. In addition to de novo replication forks, c-Myc also initiates bi-allelic replication of R2, abrogating its normal mono-allelic replication pattern, Moreover, several chromosomal regions also display c-Myc-induced illegitimate replication profiles. Thus, c-Myc can act as an illegitimate replication-licensing factor that promotes de novo replication initiation and illegitimate replication timing that adversely impacts upon genomic stability.

Here, we describe a gentle and effective method for the rapid and reproducible isolation of histone-bound extrachromosomal DNA molecules called extrachromosomal elements (EEs). This method facilitates the harvest of a specific population of EEs following their isolation from cultured cells, primary tissues, and tumor cells. Active EEs are bound to histone proteins, and these histone-bound EEs carry actively transcribing genes such as c-myc. Our method exploits the presence of histones on EEs and serves as a first-step purification procedure, allowing for the cloning or multivariant analysis of an immunopurified sample of EEs. We isolated EEs from 4-hydroxytamoxifen (4-HT)-activated Myc-ER™-regulatable Pre-B ABM cells. Following one round of immunoprecipitation, we demonstrate the purification of histone-bound EEs. We confirmed that our purification enriched for EEs that carry genes by fluorescent in situ hybridization of EEs (FISH-EEs), and we probed non-enriched and immunopurified EEs with a dihydrofolate reductase (DHFR) cDNA probe that is known to detect extrachromosomal amplification in Myc-activated cells. We demonstrate the enrichment of immunoprecipitated DHFR-containing extrachromosomal DNA molecules.

Ribonucleotide reductase is a rate-limiting enzyme in DNA synthesis and is composed of two different proteins, R1 and R2. The R2 protein appears to be rate-limiting for enzyme activity in proliferating cells, and it is phosphorylated by p34cdc2 and CDK2, mediators of cell cycle transition events. A sequence in the R2 protein at serine-20 matches a consensus sequence for p34cdc2 and CDK2 kinases. We tested the hypothesis that the serine-20 residue was the major p34cdc2 kinase site of phosphorylation. Three peptides were synthesized (from Asp-13 to Ala-28) that contained either the wild type amino acid sequence (Asp-Gln-Gln-Gln-Leu-Gln-Leu-Ser-Pro-Leu-Lys-Arg-Leu-Thr-Leu-Ala, serine peptide) or a mutation, in which the serine residue was replaced with an alanine residue (alanine peptide) or a threonine residue (threonine peptide). Only the serine peptide and threonine peptide were phosphorylated by p34cdc2 kinase. In two-dimensional phosphopeptide mapping experiments of serine peptide and Asp-N endoproteinase digested R2 protein, peptide co-migration patterns suggested that the synthetic phosphopeptide containing serine-20 was identical to the major Asp-N digested R2 phosphopeptide. To further test the hypothesis that serine-20 is the primary phosphorylated residue on R2 protein, three recombinant R2 proteins (R2-Thr, R2-Asp and R2-Ala) were generated by site-directed mutagenesis, in which the serine-20 residue was replaced with threonine, aspartic acid or alanine residues. Wild type R2 and threonine-substituted R2 proteins (R2-Thr) were phosphorylated by p34cdc2 kinase, whereas under the same experimental conditions, R2-Asp and R2-Ala phosphorylation was not detected. Furthermore, the phosphorylated amino acid residue in the R2-Thr protein was determined to be phosphothreonine. Therefore, by replacing a serine-20 residue with a threonine, the phosphorylated amino acid in R2 protein was changed to a phosphothreonine. In total, these results firmly establish that a major p34cdc2 phosphorylation site on the ribonucleotide reductase R2 protein occurs near the N-terminal end at serine-20, which is found within the sequence Ser-Pro-Leu-Lys-Arg-Leu. Comparison of ribonucleotide reductase activities between wild type and mutated forms of the R2 proteins suggested that mutation at serine-20 did not significantly affect enzyme activity.

When the Ras mitogen-activated protein kinase (MAPK) signaling pathway of quiescent cells is stimulated with growth factors or phorbol esters, the early response genes c-fos and c-myc are rapidly induced, and concurrently there is a rapid phosphorylation of histone H3, Using an antibody specific for phosphorylated Ser-10 of H3, we show that Ser-10 of H3 is phosphorylated, and we provide direct evidence that phosphorylated H3 is associated with c-fos and c-myc genes in stimulated cells. H3 phosphorylation may contribute to proto-oncogene induction by modulating chromatin structure and releasing blocks in elongation. Previously we reported that persistent stimulation of the Ras-MAPK signaling pathway in oncogene-transformed cells resulted in increased amounts of phosphorylated histone H1, Here we show that phosphorylated H3 is elevated in the oncogene-transformed mouse fibroblasts, Further we show that induction of ras expression results in a rapid increase in H3 phosphorylation. H3 phosphatase, identified as PP1, activities in ras-transformed and parental fibroblast cells were similar, suggesting that elevated H3 kinase activity was responsible for the increased level of phosphorylated H3 in the oncogene-transformed cells. Elevated levels of phosphorylated H1 and H3 may be responsible for the less condensed chromatin structure and aberrant gene expression observed in the oncogene-transformed cells.

The role of mechanical and chemical signalling pathways in the organization and function of chromatin is the subject of this review. The mechanical signalling pathway consists of the tissue matrix system that links together the three-dimensional skeletal networks, the extracellular matrix, cytoskeleton, and nuclear matrix. Intermediate filament proteins are associated with nuclear DNA, suggesting that intermediate filaments may have a role in the organization of chromatin. In human hormone-dependent breast cancer cells, the interaction between cytokeratins and chromatin is regulated by estrogens. Transcription factors, histone acetyltransferases, and histone deacetylases, which are associated with the nuclear matrix, are components of the mechanical signalling pathway. Recently, we reported that nuclear matrix-bound human and chicken histone deacetylase 1 is associated with nuclear DNA in situ, suggesting that histone deacetylase has a role in the organization of nuclear DNA. Chemical signalling pathways such as the Ras/mitogen-activated protein kinase (Ras/MAPK) pathway stimulate the activity of kinases that modify transcription factors, nonhistone chromosomal proteins, and histones. The levels of phosphorylated histones are increased in mouse fibroblasts transformed with oncogenes, the products of which stimulate the Ras/MAPK pathway. Histone phosphorylation may lead to decondensation of chromatin, resulting in aberrant gene expression.

A human Myc epitope is frequently used to tag proteins for expression experiments in nonhuman cells. We used the monoclonal 9E10 antibody specific for this epitope to analyse the expression of four proteins carrying the Myc tag in cells transfected with expression vectors. While all four proteins can be detected by immunofluorescence and immunoprecipitation assays, surprisingly, only two proteins could be detected in Western blot analysis, indicating that epitope recognition by the monoclonal antibody can be blocked in some membrane-retained ectopic proteins. Other techniques such as immunofluorescence and immunoprecipitation assays can be successfully used with the 9E10 antibody to determine potential expression of Myc-tagged proteins.

Ribonucleotide reductase, which is composed of the two protein components R1 and R2, is a highly regulated enzyme activity that is essential for DNA synthesis and repair. Recent studies have shown that elevated expression of the rate-limiting R2 component increases Raf-1 protein activation and mitogen-activated protein kinase activity and acts as a novel malignancy determinant in cooperation with H-ras and rac-1. We show that R2 cooperation in cellular transformation extends to a variety of oncogenes with different functions and cellular locations. Anchorage-independent growth of cells transformed with v-fms, v-src, A-raf, v-fes, c-myc, and ornithine decarboxylase was markedly enhanced when the R2 component of ribonucleotide reductase was overexpressed. In addition, we observed that elevated R2 expression conferred on c-myc-transformed NIH 3T3 cells an increased tumorigenic potential in immunoincompetent mice. Taken together, these observations demonstrate that the R2 protein is not only a rate-limiting component for ribonucleotide reduction but that it is also capable of acting in cooperation with a variety of oncogenes to determine transformation and tumorigenic potential.

Our recent studies have shown that deregulated expression of R2, the rate-limiting component of ribonucleotide reductase, enhances transformation and malignant potential by cooperating with activated oncogenes. We now demonstrate that the R1 component of ribonucleotide reductase has tumor-suppressing activity. Stable expression of a biologically active ectopic R1 in ras-transformed mouse fibroblast 10T(1/2) cell lines, with or without R2 overexpression, led to significantly reduced colony-forming efficiency in soft agar. The decreased anchorage independence was accompanied by markedly suppressed malignant potential in vivo. In three ras-transformed cell lines, R1 overexpression resulted in abrogation or marked suppression of tumorigenicity. In addition, the ability to form lung metastases by cells overexpressing R1 was reduced by >85%. Metastasis suppressing activity also was observed in the highly malignant mouse 10T(1/2) derived RMP-6 cell line, which was transformed by a combination of oncogenic ras, myc, and mutant p53. Furthermore, in support of the above observations with the R1 overexpressing cells, NIH 3T3 cells cotransfected with an R1 antisense sequence and oncogenic ras showed significantly increased anchorage independence as compared with control ras-transfected cells. Finally, characteristics of reduced malignant potential also were demonstrated with R1 overexpressing human colon carcinoma cells. Taken together, these results indicate that the two components of ribonucleotide reductase both are unique malignancy determinants playing opposing roles in its regulation, that there is a novel control point important in mechanisms of malignancy, which involves a balance in the levels of R1 and R2 expression, and that alterations in this balance can significantly modify transformation, tumorigenicity, and metastatic potential.

Fully thioated antisense molecules are often cytotoxic and non-specific in action. GPI2A is thioated at 7 base positions. GPI2A posses sequence-specific activity against HIV-1 gene expression and viral replication without significant cytotoxicity. Partial thioation did not compromise its uptake, cellular distribution and nuclease resistance.

Ribonucleotide reductase is a highly regulated, cell cycle-controlled activity that plays an important role in DNA synthesis and repair. Recent studies have shown that elevated expression of the rate-limiting R2 component of ribonucleotide reductase increases Raf-1 protein activation and mitogen-activated protein kinase activity and acts as a novel malignancy determinant in cooperation with activated oncogenes like H-ras. We show that hydroxyurea-resistant mouse L cells with elevated R2 gene expression and increased ribonucleotide reductase activity exhibit significantly decreased sensitivities to the chemotherapeutic compounds N-(phosphonacetyl)-L-aspartate (PALA) and methotrexate (MTX). Furthermore, BALB/c 3T3 cells containing a retroviral expression vector encoding the R2 sequence also showed decreased sensitivity to PALA and MTX when compared to cells containing the same vector but without the R2 coding region. Colonies that developed in the presence of PALA or MTX contained amplifications of the CAD or dihydrofolate reductase genes and exhibited wild-type p53 function as determined in sequence-specific p53 binding activity assays. NIH-3T3 cells containing the R2 retroviral expression vector also showed significantly decreased sensitivity to hydroxyurea and MTX but not to PALA. Furthermore, NIH-3T3 cells transfected with a vector containing the R2 sequence in antisense orientation exhibited increased sensitivity to hydroxyurea, PALA, and MTX. Similarly, mouse 10T1/2 cells that are highly transformed and drug resistant due to alterations in H-ras and a mutant oncogenic form of p53 exhibited significant increases in sensitivity to hydroxyurea, PALA, and MTX when transfected with a vector containing the R2 sequence in antisense orientation and compared to cells containing the same vector without the antisense sequence. These results indicate that altered expression of the R2 component is capable of significantly modifying drug sensitivity properties of tumor cells. We hypothesize that this occurs, at least in part, through a mechanism of increased genetic instability that is independent of direct p53 mutation or loss and involves R2 stimulation of the mitogen-activated protein kinase signal pathway.