Characterization of E2F1's oncogenic, apoptotic and tumor-suppressive activities utilizing the K5 mouse model

Though E2F1 is deregulated in most human cancers by mutations of the p16-cyclin D-Rb pathway, it also exhibits tumor suppressive activity. A transgenic mouse model overexpressing E2F1 under the control of the bovine keratin 5 (K5) promoter exhibits epidermal hyperplasia and spontaneously develops tumors in the skin and other epithelial tissues after one year of age. In a p53-deficient background, aberrant apoptosis in K5 E2F1 transgenic epidermis is reduced and tumorigenesis is accelerated. In sharp contrast, K5 E2F1 transgenic mice are resistant to papilloma formation in the DMBA/TPA two-stage carcinogenesis protocol. K5 E2F4 and K5 DP1 transgenic mice were also characterized and both display epidermal hyperplasia but do not develop spontaneous tumors even in cooperation with p53 deficiency. These transgenic mice do not have increased levels of apoptosis in their skin and are more susceptible to papilloma formation in the two-stage carcinogenesis model. These studies show that deregulated proliferation does not necessarily lead to tumor formation and that the ability to suppress skin carcinogenesis is unique to E2F1. E2F1 can also suppress skin carcinogenesis when okadaic acid is used as the tumor promoter and when a pre-initiated mouse model is used, demonstrating that E2F1's tumor suppressive activity is not specific for TPA and occurs at the promotion stage. E2F1 was thought to induce p53-dependent apoptosis through upregulation of p19ARF tumor suppressor, which inhibits mdm2-mediated p53 degradation. Consistent with in vitro studies, the overexpression of E2F1 in mouse skin results in the transcriptional activation of the p19ARF and the accumulation of p53. Inactivation of either p19ARF or p53 restores the sensitivity of K5 E2F1 transgenic mice to DMBA/TPA carcinogenesis, demonstrating that an intact p19ARF-p53 pathway is necessary for E2F1 to suppress carcinogenesis. Surprisingly, while p53 is required for E2F1 to induce apoptosis in mouse skin, p19ARF is not, and inactivation of p19ARF actually enhances E2F1-induced apoptosis and proliferation in transgenic epidermis. This indicates that ARF is important for E2F1-induced tumor suppression but not apoptosis. Senescence is another potential mechanism of tumor suppression that involves p53 and p19ARF. K5 E2F1 transgenic mice initiated with DMBA and treated with TPA show an increased number of senescence cells in their epidermis. These experiments demonstrate that E2F1's unique tumor suppressive activity in two-stage skin carcinogenesis can be genetically separated from E2F1-induced apoptosis and suggest that senescence utilizing the p19ARF-p53 pathway plays a role in tumor suppression by E2F1. ^

Induction of endoplasmic reticular stress by bortezomib: A novel therapeutic strategy for pancreatic cancer

Bortezomib (VELCADE™, formerly known as PS-341) is a selective and potent inhibitor of the proteasome that was recently FDA-approved for the treatment of multiple myeloma. Despite its success in multiple myeloma and progression into clinical trials for other malignancies, bortezomib's exact mechanism of action remains undefined. The major objective of this study was to evaluate the anticancer activity of this drug using in vitro and in vivo pancreatic cancer models and determine whether bortezomib-induced apoptosis occurs via induction of endoplasmic reticular (ER) stress. The investigation revealed that bortezomib inhibited tumor cell proliferation via abrogation of cdk activity and induced apoptosis in pancreatic cancer cell lines. I hypothesized that bortezomib-induced apoptosis was triggered by a large accumulation ubiquitin-conjugated proteins that resulted in ER stress. My data demonstrated that bortezomib induced a unique type of ER stress in that it inhibited PKR-like ER kinase (PERK) and subsequent phosphorylation of eukaryotic initiation factor 2α (eif2α), a key event in translational suppression. The combined effects of proteasome inhibition and the failure to attenuate translation resulted in an accumulation of aggregated proteins (proteotoxicity), JNK activation, cytochrome c release, caspase-3 activation, and DNA fragmentation. Bortezomib also enhanced apoptosis induced by other agents that stimulated the unfolded protein response (UPR), demonstrating that translational suppression is a critical cytoprotective mechanism during ER stress. Tumor cells attempt to survive bortezomib-induced ER stress by sequestering aggregated proteins into large structures, termed aggresomes. Since histone deacetylase 6 (HDAC6) is essential for aggresome formation, tumor cells may be sensitized to bortezomib-induced apoptosis by blocking HDAC function. My results demonstrated that HDAC inhibitors disrupted aggresome formation and synergized with bortezomib to induce apoptosis in pancreatic cancer or multiple myeloma cells in vitro and in orthotopic pancreatic tumors in vivo. Taken together, my data establish a mechanistic link between bortezomib-induced aggresome formation, ER stress, and apoptosis and identify a novel therapeutic strategy for the treatment of pancreatic cancer and other hematologic and solid malignancies. ^

Mechanisms involved in suppression of the elicitation of immune responses by ultraviolet light

The ultraviolet radiation (UVR) present in sunlight is the primary cause of nonmelanoma skin cancer and has been implicated in the development of cutaneous malignant melanoma. Ultraviolet radiation also suppresses the immune response. In the majority of studies investigating the mechanisms regulating UV-induced immune suppression, UV is used to suppress the induction of immune responses. Equally important, is the ability of UVR to suppress established immune responses, such as the recall reaction in humans, which protects against microbial infections. We established a murine model to help elucidate the immunological mechanisms governing UV-induced suppression of the elicitation of immune responses. 80 kJ/m2 of UVR nine days after sensitization consistently suppressed the elicitation of delayed type hypersensitivity reaction to C. albicans . We found ultraviolet A (320±400 nm) radiation was as effective as solar-simulated ultraviolet A + B (290±400 nm) in suppressing the elicitation of an established immune response. The mechanisms involved in UV-induced suppression of the induction & elicitation of the immune response are similar. For example, mice irradiated with UV after immunization generated antigen-specific T suppressor cells. Injection of monoclonal antibodies to IL-10 or recombinant IL-12 immediately after exposure to UVR blocked immune suppression. Liposomes containing bacteriophage T4N5 to the skin of mice also prevented immune suppression, demonstrating an essential role for ultraviolet-induced DNA damage in the suppression of established immune reactions. ^ In addition to damaging DNA, UV initiates immune suppression through the isomerization of urocanic acid in the epidermis. Here we provide evidence that cis-UCA induces systemic immunosuppression via the serotonin (5-hydroxyyryptamine; 5-HT) receptor. Biochemical and immunological analysis indicate that cis-UCA binds to, and activates, the serotonin receptor. Moreover, serotonin specific antibodies block UV- and/or cis-UCA-induced immune suppression. Our findings identify cis-UCA as novel serotonin receptor ligand and indicate that serotonin receptor engagement can activate immune suppression. Cumulatively, our data suggest that similar immune regulatory mechanisms are activated regardless of whether we expose mice to solar-simulated UV (UVA + UVB) radiation or UVA only, and that ultraviolet radiation activates similar immunologic pathways to suppress the induction or the elicitation of the immune response. ^

Nitric oxide contributes to LPS/IFN-gamma-induced macrophage apoptosis via JNK and BCL-2 family regulation

Lipopolysaccharide (LPS) and interferon-gamma (IFN) activate macrophages and produce nitric oxide (NO) by initiating the expression of inducible Nitric Oxide Synthase (iNOS). Prolonged LPS/IFN-activation results in the death of macrophage-like RAW 264.7 cells and wild-type murine macrophages. This study was implemented to determine how NO contributes to LPS/IFN-induced macrophage death. The iNOS-specific inhibitor L-NIL protected RAW 264.7 cells from LPS/IFN-activated death, supporting a role for NO in the death of LPS/IFN-activated macrophages. A role for iNOS in cell death was confirmed in iNOS-/- macrophages which were resistant to LPS/IFN-induced death. Cell death was accompanied by nuclear condensation, caspase 3 activation, and PARP cleavage, all of which are hallmarks of apoptosis. The involvement of NO in modulating the stress-activated protein kinase (SAPK)/c-jun N-terminal kinase (JNK) signal transduction pathway was examined as a possible mechanism of LPS/IFN-mediated apoptosis. Western analysis demonstrated that NO modifies the phosphorylation profile of JNK and promotes activation of JNK in the mitochondria in RAW 264.7 cells. Inhibition of JNK with sIRNA significantly reduced cell death in RAW 264.7 cells, indicating the participation of the JNK pathway in LPS/IFN-mediated death. JNK has been demonstrated to induce mitochondrial-mediated apoptosis through modulation of Bcl-2 family members. Therefore, the effect of NO on the balance between pro- and anti-apoptotic Bcl-2 family members was examined. In RAW 264.7 cells, Bim was upregulated and phosphorylated by LPS/IFN independently of NO. However, co-immunoprecipitation studies demonstrated that NO promotes the association of Bax with the BimL splice variant. Examination of Bax phosphorylation by metabolic labeling demonstrated that Bax is basally phosphorylated and becomes dephosphorylated upon LPS/IFN treatment. L-NIL inhibited the dephosphorylation of Bax, indicating that Bax dephosphorylation is NO-dependent. NO also mediated LPS/IFN-induced downregulation of Mcl-1, an anti-apoptotic Bcl-2 family member, as demonstrated by Western blotting for Mcl-1 protein expression. Thus, NO contributes to macrophage apoptosis via a JNK-mediated mechanism involving interaction between Bax and Bim, dephosphorylation of Bax, and downregulation of Mcl-1. ^

Examination of the engraftment of exogenous mesenchymal stem cells in ovarian tumors and their potential use as delivery vehicles for therapeutic genes

Interactions between neoplastic cells and the host stroma play a role in both tumor cell migration and proliferation. Stromal cells provide structural support for malignant cells, modulate the tumor microenvironment, and influence phenotypic behavior as well as the aggressiveness of the malignancy. In response, the tumor provides growth factors, cytokines, and cellular signals that continually initiate new stromal reactions and recruit new cells into the microenvironment to further support tumor growth. Since growing tumors recruit local cells, as well as supplemental cells from the circulation, such as fibroblasts and endothelial precursors, the question arises if it would be possible to access circulating stromal cells to modify the tumor microenvironment for therapeutic benefits. One such cell type, mesenchymal stem cells (MSC), could theoretically be engrafted into stroma. MSC are pluripotent cells that have been shown to form stromal elements such as myofibroblasts, perivascular tissues and connective tissues. Several reports have demonstrated that MSC can incorporate into sites of wound healing and tissue repair, due to active tissue remodeling and local paracrine factors, and given the similarity between wound healing and the carcinoma induced stromal response one can hypothesize that MSC have the potential to be recruited to sites of tumor development. In addition, gene-modified MSC could be used as cellular vehicles to deliver gene products into tumors. My results indicate that MSC home to and participate in tumor stroma formation in ovarian tumor xenografts in mice. Additionally, once homed to tumor beds, MSC proliferate rapidly and integrate. My studies aim at understanding the fate of MSC in the tumor microenvironment, as well as utilizing them for cellular delivery of therapeutic genes into the stroma of ovarian carcinomas. ^

Regulation of RNA splicing by nonsense mutations

Translation termination as a result of premature nonsense codon-incorporation in a RNA transcript can lead to the production of aberrant proteins with gain-of-function or dominant negative properties that could have deletrious effects on the cell. T-cell Receptor (TCR) genes acquire premature termination codons two-thirds of the time as a result of the error-prone programmed rearrangement events that normally occur during T-cell development. My studies have focused on the fate of TCR precursor mRNAs in response to in-frame nonsense mutations. ^ Previous published studies from our laboratory have shown that TCR precursor mRNAs are subject to nonsense mediated upregulation of pre-mRNA (NMUP). In this dissertation, I performed substitution and deletion analysis to characterize specific regions of TCR which are required to elicit NMUP. I performed frame- and factor-dependence studies to determine its relationship with other nonsense codon induced responses using several approaches including (i) translation dependence studies (ii) deletion and mutational analysis, as well as (iii) siRNA mediated knockdown of proteins involved. I also addressed the underlying molecular mechanism for this pre-mRNA upregulation by (i) RNA half-life studies using a c-fos inducible promoter, and (ii) a variety of assays to determine pre-mRNA splicing efficiency. ^ Using these approaches, I have identified a region of TCR that is both necessary and sufficient to elicit (NMUP). I have also found that neither cytoplasmic translation machinery nor the protein UPF1 are involved in eliciting this nuclear event. I have shown that the NMUP can be induced not only by nonsense and frameshift mutations, but also missense mutations that disrupt a cis splicing element in the exon that contains the mutation. However, the effect of nonsense mutations on pre-mRNA is unique and distinguishable from that of missense mutations in that nonsense mutations can upregulate pre-mRNA in a frame-dependent manner. Lastly, I provide evidence that NMUP occurs by a mechanism in which nonsense mutations inhibit the splicing of introns. In summary, I have found that TCR precursor mRNAs are subject to multiple forces involving both RNA splicing and translation that can either increase or decrease the levels of these precursor mRNAs. ^

A systematic analysis of INO80 in DNA replication

ATP-dependent chromatin remodeling has been shown to be critical for transcription and DNA repair. However, the involvement of ATP-dependent chromatin remodeling in DNA replication remains poorly defined. Interestingly, we found that the INO80 chromatin-remodeling complex is directly involved in the DNA damage tolerance pathways activated during DNA replication. DNA damage tolerance is important for genomic stability and is controlled by formation of either mono-ubiquitinated or multi-ubiquitinated PCNA, which respectively induce error prone or error-free replication bypass of the lesions. In addition, homologous recombination (HR) mediated by the Rad51 pathway is also involved in the DNA damage tolerance pathways. ^ We found that INO80 is specifically recruited to replication origins during S phase in a genome-wide fashion. In addition, DNA combing analysis shows INO80 is required for the resumption of replication at stalled forks induced by methyl methane-sulfonate (MMS). Mechanistically, we find that INO80 is required for PCNA ubiquitination as well as for Rad51 mediated processing of replication forks after MMS treatment. Furthermore, chromatin immunoprecipitation at specific ARSs indicates INO80 is necessary for Rad18 and Rad51 recruitment to replication forks after MMS treatment. Moreover, 2D gel analysis shows INO80 is necessary to process Rad51 mediated intermediates at impeded replication forks. ^ In conclusion, our findings establish a novel role of a chromatin-remodeling complex in DNA damage tolerance pathways and suggest that chromatin remodeling is fundamentally important to ensure faithful replication of DNA and genome stability in eukaryotes. ^

Identification of a conserved cluster in the RH domain of GRK critical for activation by GPCRs

One of the most critical aspects of G Protein Coupled Receptors (GPCRs) regulation is their rapid and acute desensitization following agonist stimulation. Phosphorylation of these receptors by GPCR kinases (GRK) is a major mechanism of desensitization. Considerable evidence from studies of rhodopsin kinase and GRK2 suggests there is an allosteric docking site for the receptor distinct from the GRK catalytic site. While the agonist-activated GPCR appears crucial for GRK activation, the molecular details of this interaction remain unclear. Recent studies suggested an important role for the N- and C-termini and domains in the small lobe of the kinase domain in allosteric activation; however, neither the mechanism of action of that site nor the RH domain contributions have been elucidated. To search for the allosteric site, we first indentified evolutionarily conserved sites within the RH and kinase domains presumably deterministic of protein function employing evolutionary trace (ET) methodology and crystal structures of GRK6. Focusing on a conserved cluster centered on helices 3, 9, and 10 in the RH domain, key residues of GRK5 and 6 were targeted for mutagenesis and functional assays. We found that a number of double mutations within helices 3, 9, and 10 and the N-terminus markedly reduced (50–90%) the constitutive phosphorylation of the β-2 Adrenergic Receptor (β2AR) in intact cells and phosphorylation of light-activated rhodopsin (Rho*) in vitro as compared to wild type (WT) GRK5 or 6. Based on these results, we designed peptide mimetics of GRK5 helix 9 both computationally and through chemical modifications with the goal of both confirming the importance of helix 9 and developing a useful inhibitor to disrupt the GPCR-GRK interaction. Several peptides were found to block Rho* phosphorylation by GRK5 including the native helix 9 sequence, Peptide Builder designed-peptide preserving only the key ET residues, and chemically locked helices. Most peptidomimetics showed inhibition of GRK5 activity greater than 80 % with an IC50 of ∼ 30 µM. Alanine scanning of helix 9 has further revealed both essential and non-essential residues for inhibition. Importantly, substitution of Arg 169 by an alanine in the native helix 9-based peptide gave an almost complete inhibition at 30 µM with an IC50 of ∼ 10 µM. In summary we report a previously unrecognized crucial role for the RH domain of GRK5 and 6, and the subsequent identification of a lead peptide inhibitor of protein-protein interaction with potential for specific blockade of GPCR desensitization. ^

E2F1's role in ultraviolet-induced DNA damage response

The E2F1 transcription factor is a well-known regulator of cell proliferation and apoptosis, but its role in the DNA damage response is less clear. It has been shown that E2F1 becomes stabilized in response to DNA double strand breaks (DSBs) and accumulates at sites of DSBs. This process requires ATM kinase and serine 31 phosphorylation, which provides a binding site for TopBp1. However, the role of E2F1 at sites of DNA damage is not clear. We expanded the study of E2F1's role in the DNA damage response by exploring its functions in ultraviolet (UV) induced DNA damage, and identified that E2F1 promotes DNA repair and cell survival. To further investigate the mechanisms underlying our findings, we examined the possibility for direct involvement of E2F1 in DNA repair. We found that E2F1 localizes to sites of UV irradiation-induced DNA damage dependent on the ATR kinase and serine 31 of E2F1. E2F1 also associates with the GCN5 histone acetyltransferase in response to UV irradiation and recruits GCN5 to sites of DNA damage. This correlates with an increase in histone H3 lysine 9 (H3K9) acetylation and chromatin relaxation. In the absence of E2F1 or GCN5, nucleotide excision repair (NER) proteins do not efficiently localize to sites of UV damage and DNA repair is impaired. E2F1 mutants unable to bind DNA or activate transcription retain the ability to stimulate NER. These findings demonstrate a non-transcriptional role for E2F1 in DNA repair involving GCN5-mediated H3K9 acetylation and increased accessibility to the NER machinery. ^

Expression of p53, {\it * ras\/} and PCNA in human colonic neoplasms: Possible biomarkers of malignant potential

Colorectal cancer is a leading cause of cancer mortality and early detection can significantly improve the clinical outcome. Most colorectal cancers arise from benign neoplastic lesions recognized as adenomas. Only a small percentage of all adenomas will become malignant. Thus, there is a need to identify specific markers of malignant potential. Studies at the molecular level have demonstrated an accumulation of genetic alterations, some hereditary but for the most occurring in somatic cells. The most common are the activation of ras, an oncogene involved in signal transduction, and the inactivation of p53, a tumor suppressor gene implicated in cell cycle regulation. In this study, 38 carcinomas, 95 adenomas and 20 benign polyps were analyzed by immunohistochemistry for the abnormal expression of p53 and ras proteins. An index of cellular proliferation was also measured by labeling with PCNA. A general overexpression of p53 was immunodetected in 66% of the carcinomas, while 26% of adenomas displayed scattered individual positive cells or a focal high concentration of positive cells. This later was more associated with severe dysplasia. Ras protein was detected in 37% of carcinomas and 32% of adenomas mostly throughout the tissue. p53 immunodetection was more frequent in adenomas originating in colons with synchronous carcinomas, particularly in patients with familial adenomatous polyposis and it may be a useful marker in these cases. Difference in the frequency of p53 and ras alterationbs was related to the location of the neoplasm. Immunodetection of p53 protein was correlated to the presence of a mutation in p53 gene at exon 7 and 5 in 4/6 carcinomas studied and 2 villous adenomas. Thus, we characterized in adenomas the abnormal expression of two proteins encoded by the most commonly altered genes in colorectal cancer. p53 alteration appears to be more specifically associated with transition to malignancy than ras. By using immunohistochemistry, a technique that keeps the architecture of the tissue intact, it was possible to correlate these alterations to histopathological characteristics that were associated with higher risks for transformation: villous content, dysplasia and size of adenoma. ^

Metabolism and actions of 2 -chloro-2$\sp\prime$-fluoro-arabinosyladenine

2-Chloro-9-(2-deoxy-2-fluoro-$\beta $-D-arabinofuranosyl)adenine(Cl-F-ara-A) is a new deoxyadenosine analogue which is resistant to phosphorolytic cleavage and deamination, and exhibits therapeutic activity for both leukemia and solid tumors in experimental systems. To characterize its mechanism of cytotoxicity, the present study investigated the cellular pharmacology and the biochemical and molecular mechanisms of action of Cl-F-ara-A, from entrance of the drug into the cell, chemical changes to active metabolites, targeting on different cellular enzymes, to final programmed cell death response to the drug treatment.^ Cl-F-ara-A exhibited potent inhibitory action on DNA synthesis in a concentration-dependent and irreversible manner. The mono-, di-, and triphosphates of Cl-F-ara-A accumulated in cells, and their elimination was non-linear with a prolonged terminal phase, which resulted in prolonged dNTP depression. Ribonucleotide reductase activity was inversely correlated with the cellular Cl-F-ara-ATP level, and the inhibition of the reductase was saturated at higher cellular Cl-F-ara-ATP concentrations. The sustained inhibition of ribonucleotide reductase and the consequent depletion of deoxynucleotide triphosphate pools result in a cellular Cl-F-ara-ATP to dATP ratio which favors analogue incorporation into DNA.^ Incubation of CCRF-CEM cells with Cl-F-ara-A resulted in the incorporation of Cl-F-ara-AMP into DNA. A much lesser amount was associated with RNA, suggesting that Cl-F-ara-A is a more DNA-directed compound. The site of Cl-F-ara-AMP in DNA was related to the ratio of the cellular concentrations of the analogue triphosphate and the natural substrate dATP. Clonogenicity assays showed a strong inverse correlation between cell survival and Cl-F-ara-AMP incorporation into DNA, suggesting that the incorporation of Cl-F-ara-A monophosphate into DNA is critical for the cytotoxicity of Cl-F-ara-A.^ Cl-F-ara-ATP competed with dATP for incorporation into the A-site of the extending DNA strand catalyzed by both DNA polymerase $\alpha$ and $\varepsilon$. The incorporation of Cl-F-ara-AMP into DNA resulted in termination of DNA strand elongation, with the most pronounced effect being observed at Cl-F-ara-ATP:dATP ratio $>$1. The presence of Cl-F-ara-AMP at the 3$\sp\prime$-terminus of DNA also resulted in an increased incidence of nucleotide misincorporation in the following nucleotide position. The DNA termination and the nucleotide misincorporation induced by the incorporation of Cl-F-ara-AMP into DNA may contribute to the cytotoxicity of Cl-F-ara-A. ^

Liposome -mediated delivery of all-{\it trans\/}-retinoic acid

Because of its antiproliferative and differentiation-inducing properties, all-trans-retinoic acid (ATRA) has been used as a chemopreventive and therapeutic agent, for treatment various cancers including squamous cell carcinomas (SCCs). Long-term treatment with ATRA is associated with toxic effects in patients leading to acute or chronic hypervitaminosis syndrome. Moreover, prolonged treatment with oral ATRA leads to acquired resistance to the differentiation-inducing effects of the drug. This resistance is attributed to the induction of cytochrome P-450-dependent catabolic enzymes that lead to accelerated ATRA metabolism and decline in circulating levels. Most of these problems could be circumvented by incorporating ATRA in liposomes (L-ATRA) which results in sustained drug release, decrease in drug-associated toxicity, and protection of the drug from metabolism in the host. Liposomes also function as a solubilization matrix enabling lipophilic drugs like ATRA to be aerosolized and delivered directly to target areas in the aerodigestive tract and lungs. Of the 14 formulations tested, the positively-charged liposome, DPPC:SA (9:1, w/w) was found to be most effective in interacting with SCC cell lines. This, L-ATRA formulation was stable in the presence of serum proteins and buffered the toxic effects of the drug against several normal and malignant cell lines. The positive charge attributed by the presence of SA was critical for increased uptake and retention of L-ATRA by SCC cell lines and tumor spheroids. L-ATRA was highly effective in mediating differentiation in normal and transformed epithelial cells. Moreover, liposomal incorporation significantly reduced the rate of ATRA metabolism by cells and isolated liver microsomes. In vivo studies revealed that aerosol delivery is an effective way of administering L-ATRA, in terms of its safety and retention by lung tissue. The drug so delivered, is biologically active and had no toxic effects in mice. From these results, we conclude that liposome-incorporation is an excellent way of delivering ATRA to target tissues. The results obtained may have important clinical implications in treating patients with SCCs of the aerodigestive tract. ^

Mechanism of silica -induced apoptosis in human alveolar macrophages

The mechanisms involved in the development of pulmonary silicosis have not been well defined, however most current evidence implicates a central role for alveolar macrophages in this process. We propose that the fibrotic potential of a particulate depends upon its ability to cause apoptosis in alveolar macrophage (AM). The overall goal of this study was to determine the mechanism of silica-induced apoptosis of AM. Human AM were treated with fibrogenic, poorly fibrogenic and nonfibrogenic model particulates, such as, silica, amorphous silica and titanium dioxide, respectively (equal surface area). Treatment with silica resulted in apoptosis in human AM as observed by morphology, DNA fragmentation and Cell Death ELISA assays. In contrast, amorphous silica and titanium dioxide demonstrated no significant apoptotic potential. To elucidate the possible mechanism by which silica causes apoptosis, we investigated the role of the scavenger receptor (SR) in silica-induced apoptosis. Cells were pretreated with and without SR ligand binding inhibitors, polyinosinic acid (Poly I), fucoidan and high density lipoprotein (HDL), prior to silica treatment. Pretreatment with Poly I and fucoidan resulted in significant inhibition of silica-induced apoptosis suggesting that silica-induced AM apoptosis is mediated via the SR. Further, we examined the involvement of interleukin converting enzyme (ICE) family of proteases in silica-mediated apoptosis. Silica activated ICE, Ich-1L, cpp32 beta and cleavage of PARP. Taken together, these results suggested that (1) fibrogenic particulates, such as, silica caused apoptosis of alveolar macrophages, (2) this apoptotic potential of fibrogenic particulates may be a critical factor in initiating an inflammatory response resulting in fibrosis, (3) silica-induced apoptosis of alveolar macrophages may be due to the interaction of silica particulates with the SR, and (4) silica-induced apoptosis involves the activation of the ICE family of proteases. An understanding of the molecular events involved in fibrogenic particulate-induced apoptosis may provide a useful insight into the mechanism involved in particulate-induced fibrosis. ^

Experimental models for p53, hepatitis B and aflatoxin hepatocarcinogenesis

The major risk factors for liver cancer in Southeast Asia: HBV infection, aflatoxin exposure and p53 expression/mutation, were examined in experimental models. Four groups were examined for development of hepatocellular carcinoma (HCC) with and without neonatal exposure to aflatoxin (AFB$\sb1)$: (Group I.) Transgenic HBsAg mice with one p53 allele. (Group II) Transgenic HBsAg mice with two p53 alleles. (Group III) Non-transgenic litter mates with one p53 allele. (Group IV) Non-transgenic litter mates with two p53 alleles. HCC developed in Group I animals exposed to aflatoxin at an earlier time and were of a higher grade than those seen later in other groups. These results provide an explanation for as to why p53 is a target for deletion and/or mutation in human HCC especially when found in high risk areas where HBV infection and Aflatoxin B1 food contamination is high, and nicely illustrates a synergistic interaction among these three factors. None of the tumors analyzed had loss or mutation in the p53 gene.^ To determine the significance of the specific p53ser249 mutation found in HBV/aflatoxin associated human hepatomas in an in-vivo experimental model using transgenic mice, a two-nucleotide change in the mouse p53 gene at amino acid position 246, which is equivalent to that of 249 in human p53, was introduced. Transgenic mice with mutant p53 controlled by the albumin promoter were generated and shown to express the p53ser246 mutant RNA and protein specifically in liver. Three groups were examined for development of HCC with and without neonatal exposure to aflatoxin: (Group V) Transgenic p53ser246 mice with two p53 alleles. (Group VI) Transgenic p53ser246 mice with one p53 allele. (Group VII) Double transgenic for p53ser246 and HBsAg with two p53 alleles. One hundred percent of male mice with the three risk factors injected with aflatoxin developed high grade liver tumors, compared to 66.6% from group VI and only 14.2% of group V suggesting synergistic interaction between HBsAg and this particular ser246 p53 mutation.^ In order to examine the growth properties of hepatocytes and correlation with p53 loss and/or mutation, cell proliferation and ploidy analysis of liver from normal heterozyous, homozygous null mice and from transgenic mutant p53ser246, mice were studied. Loss of wild-type p53 increased G1/G0 ratios of cells as well as proliferation and decreased cell ploidy. The mutant p53ser246 did not show a significant effect on cell ploidy or proliferation. However a striking 5-10X increase in G1/G0 ratio suggests that this specific mutation specifically induces G0 to G1 transition, which in turn further predisposes hepatocytes to the damaging effect of Aflatoxin. (Abstract shortened by UMI.) ^

Genome instability quantified in constitutive tissues as well as putatively stable tumor tissue in hereditary non -polyposis colon cancer patients by small pool PCR

Microsatellite instability (MSI) is a hallmark of the mutator phenotype associated with Hereditary Non-Polyposis Colon Cancer (HNPCC). The MSI-High (MSI-H) HNPCC population has been well characterized, but the microsatellite low and stable (MSI-L/MSS) HNPCC population is much less understood. We hypothesize there are significant levels of MSI in HNPCC DNA classified as MSI-L/MSS, but no single variant allele makes up a sufficient population in the tumor DNA to be detected by standard analysis. Finding variants would suggest there is a mutator phenotype for the MSI-L/MSS HNPCC population that is distinct from the MSI-H HNPCC populations. This study quantified and compared MSI in HNPCC patients previously shown to be MSI-H, MSI-L/MSS and an MSI-H older, sporadic colorectal cancer patient. Small-pool Polymerase Chain Reactions (SP-PCRs) were conducted where the DNAs from each sample and controls are diluted into multiple pools, each containing approximately single genome equivalents. At least 100 alleles/sample were studied at six microsatellite loci. Mutant fragments were identified, quantified, and compared using Poisson statistics. Most of the variants were small deletions or insertions, with more mutants being deletions, as has been previously described in yeast and transgenic mice. SP-PCR, where most of the pools contained only 3 or less fragments, enabled identification of variants too infrequent to be detected by large pool PCR. Mutant fragments in positive control MSI-H tumor samples ranged from 0.26 to 0.68 in at least 4 of the 6 loci tested and were consistent with their MSI-H status. In the so called MSS tumors and constitutive tissues (normal colon tissue, and PBLs) of all the HNPCC patients, low, but significant levels of MSI were seen in at least two of the loci studied. This phenomenon was not seen in the sporadic MSI constitutive tissues nor the normal controls and suggests haploinsufficiency, gain-of-function, or a dominant/negative basis of the instability in HNPCC patients carrying germline mutations for tumor suppressor genes. A different frequency and spectrum of mutant fragments suggests a different genetic basis (other than a major mutation in MLH1 or MSH2) for disease in MSI-L and MSS HNPCC patients. ^