Marizomib, a proteasome inhibitor for all seasons: preclinical profile and a framework for clinical trials.

The proteasome has emerged as an important clinically relevant target for the treatment of hematologic malignancies. Since the Food and Drug Administration approved the first-in-class proteasome inhibitor bortezomib (Velcade) for the treatment of relapsed/refractory multiple myeloma (MM) and mantle cell lymphoma, it has become clear that new inhibitors are needed that have a better therapeutic ratio, can overcome inherent and acquired bortezomib resistance and exhibit broader anti-cancer activities. Marizomib (NPI-0052; salinosporamide A) is a structurally and pharmacologically unique ß-lactone-γ-lactam proteasome inhibitor that may fulfill these unmet needs. The potent and sustained inhibition of all three proteolytic activities of the proteasome by marizomib has inspired extensive preclinical evaluation in a variety of hematologic and solid tumor models, where it is efficacious as a single agent and in combination with biologics, chemotherapeutics and targeted therapeutic agents. Specifically, marizomib has been evaluated in models for multiple myeloma, mantle cell lymphoma, Waldenstrom's macroglobulinemia, chronic and acute lymphocytic leukemia, as well as glioma, colorectal and pancreatic cancer models, and has exhibited synergistic activities in tumor models in combination with bortezomib, the immunomodulatory agent lenalidomide (Revlimid), and various histone deacetylase inhibitors. These and other studies provided the framework for ongoing clinical trials in patients with MM, lymphomas, leukemias and solid tumors, including those who have failed bortezomib treatment, as well as in patients with diagnoses where other proteasome inhibitors have not demonstrated significant efficacy. This review captures the remarkable translational studies and contributions from many collaborators that have advanced marizomib from seabed to bench to bedside.

Proteasome inhibitors activate autophagy as a cytoprotective response in human prostate cancer cells.

The ubiquitin-proteasome and lysosome-autophagy pathways are the two major intracellular protein degradation systems that work cooperatively to maintain homeostasis. Proteasome inhibitors (PIs) have clinical activity in hematological tumors, and inhibitors of autophagy are also being evaluated as potential antitumor therapies. In this study, we found that chemical PIs and small interfering RNA-mediated knockdown of the proteasome's enzymatic subunits promoted autophagosome formation, stimulated autophagic flux, and upregulated expression of the autophagy-specific genes (ATGs) (ATG5 and ATG7) in some human prostate cancer cells and immortalized mouse embryonic fibroblasts (MEFs). Upregulation of ATG5 and ATG7 only occurred in cells displaying PI-induced phosphorylation of the eukaryotic translation initiation factor 2 alpha (eIF2alpha), an important component of the unfolded protein responses. Furthermore, PIs did not induce autophagy or upregulate ATG5 in MEFs expressing a phosphorylation-deficient mutant form of eIF2alpha. Combined inhibition of autophagy and the proteasome induced an accumulation of intracellular protein aggregates reminiscent of neuronal inclusion bodies and caused more cancer cell death than blocking either degradation pathway alone. Overall, our data show that proteasome inhibition activates autophagy through a phospho-eIF2alpha-dependent mechanism to eliminate protein aggregates and alleviate proteotoxic stress.

Measuring soluble forms of extracellular cytokeratin 18 identifies both apoptotic and necrotic mechanisms of cell death produced by adenoviral-mediated interferon alpha: possible use as a surrogate marker.

Adenoviral transduction of human bladder cancer cells with human interferon alpha-2b (Ad-IFN) produces cancer-specific cell death through direct and indirect mechanisms. The indirect mechanisms involve the secreted IFN produced, which kill, IFN protein-sensitive cancer cells, as well as yet unidentified bystander factors, which are cytotoxic to neighboring cancer cells. The direct cell kill results from transfection and expression of Ad-IFN in the cancer cells. As the molecular forms of cytokeratin 18, either caspase cleaved or not, have been associated with apoptotic or necrotic cell death, respectively, we determined if increases in either or both cytokeratin 18 forms could be observed following IFNalpha protein or Ad-IFN treatment of bladder carcinoma cells. Quantification of M30 and M65 enzyme-linked immunosorbent assays (assays for cytokeratin 18 associated apoptotic and necrotic cell death, respectively) were used as surrogate markers of the cell death produced. In the IFN protein-sensitive RT4 bladder cancer cells, IFN produced primarily M30-related cell death, whereas Ad-IFN treatment resulted in high levels of both M30 and M65. In contrast, conditioned medium from Ad-IFN-treated cells whether from normal human urothelial cells or bladder cancer cells caused increases mainly in M30 levels when added to IFN protein resistant KU7 or UC9 bladder cancer cells, suggesting that the bystander factors present in the conditioned medium produced primarily apoptotic cell death. In addition, a significant increase in M65 levels above that observed for M30 was seen when the IFN protein resistant KU7 and UC9 cells were treated with Ad-IFN, again indicating there is additional necrotic-related cell death produced by Ad-IFN as well. Normal urothelial cells showed no cytotoxicity nor increases in M30 or M65 after Ad-IFN treatment. As intravesical Ad-IFN treatment is presently being evaluated for its efficacy in superficial bladder cancer measurement of M30 and M65 levels in the urine at various time points before and after Ad-IFN treatment may provide not only a biomarker of efficacy but also evidence for the different types and proportion of cell kill produced by the various mechanisms of cell kill in the tumors of individual patients.

Early RB94-produced cytotoxicity in cancer cells is independent of caspase activation or 50 kb DNA fragmentation.

RB94, which lacks the N-terminal 112 amino-acid residues of the full-length retinoblastoma protein (RB110) is a more potent inhibitor of cancer cell growth than RB110, being cytotoxic to all cancer cell lines studied, independent of their genetic abnormalities. Although we initially thought RB94-induced cell death was caspase-dependent, such caspase activation now appears to be a late event. Cells that remained attached 48 h after transduction with Ad-RB94 showed, among other changes, nuclear enlargement, peripheral nuclear chromatin condensation and often micronucleation. In addition, the cells were TdT-mediated dUTP nick end labeling (TUNEL) positive but showed no cleavage of caspase 3 or 9. Only after the cells detached was cleavage of both caspase 3 and 9 observed. These TUNEL-positive cells showed neither cytochrome c mitochondrial translocation usually found in typical apoptotic cells nor DNA laddering indicative of oligonucleosomal DNA fragmentation. In addition, although 50 kb DNA fragmentation was produced in these TUNEL-positive cells, which was dependent on apoptosis-inducing factor (AIF), inhibiting this fragmentation by siAIF did not inhibit TUNEL formation or cytotoxicity. As RB94 will soon be used for gene therapy further understanding the molecular basis of these early changes in killing cancer cells is one of our particularly important present goals.

Phase II trial of imatinib mesylate in patients with metastatic melanoma.

Metastatic melanoma cells express a number of protein tyrosine kinases (PTKs) that are considered to be targets for imatinib. We conducted a phase II trial of imatinib in patients with metastatic melanoma expressing at least one of these PTKs. Twenty-one patients whose tumours expressed at least one PTK (c-kit, platelet-derived growth factor receptors, c-abl, or abl-related gene) were treated with 400 mg of imatinib twice daily. One patient with metastatic acral lentiginous melanoma, containing the highest c-kit expression among all patients, had dramatic improvement on positron emission tomographic scan at 6 weeks and had a partial response lasting 12.8 months. The responder had a substantial increase in tumour and endothelial cell apoptosis at 2 weeks of treatment. Imatinib was fairly well tolerated: no patient required treatment discontinuation because of toxicity. Fatigue and oedema were the only grade 3 or 4 toxicities that occurred in more than 10% of the patients. Imatinib at the studied dose had minimal clinical efficacy as a single-agent therapy for metastatic melanoma. However, based on the characteristics of the responding tumour in our study, clinical activity of imatinib, specifically in patients with melanoma with certain c-kit aberrations, should be examined.

Adenoviral-mediated interferon alpha overcomes resistance to the interferon protein in various cancer types and has marked bystander effects.

We have previously shown that intravesical administration of adenovirus encoding human interferon alpha-2b (Ad-IFN) induced a marked regression of superficial human bladder tumors derived from cells that are resistant to over 1 million units/ml of IFNalpha protein in vitro. In addition, Ad-IFN appeared to produce strong bystander effects. In this study, we show that Ad-IFN causes marked inhibition of cell growth and apoptosis in cells of various tumor types, all of which are resistant to IFNalpha protein. In addition, strong perinuclear IFN staining was seen in all cell lines following Ad-IFN transfection and was never observed after exposure to the IFN protein. Ad-IFN induced proteolytic processing of caspases 3, 8 and 9, indicative of enzymatic activation. However, the caspase-8-selective inhibitor, IETDfmk, blocked apoptosis only in the cell lines that were sensitive to the IFNalpha protein and had minimal effect on Ad-IFN-induced caspase-3 or -9 processing and cell death, indicating that death receptor-independent mechanism(s) were involved in the cytotoxic effects observed for cancer cell lines resistant to the IFNalpha protein. Moreover, we document that a yet to be identified soluble factor(s) is responsible for causing the bystander effect observed following Ad-IFN treatment in IFN protein-resistant cancer cells.

Increased mitochondrial biogenesis in primary leukemia cells: the role of endogenous nitric oxide and impact on sensitivity to fludarabine.

B cell chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia in the Western hemisphere, yet many biological and molecular features of the disease remain undefined. CLL cells generate increased levels of radical species such as superoxide and nitric oxide (NO), which is associated with mitochondrial DNA mutations. Considering that NO levels can affect mitochondrial biogenesis, we hypothesized that the inherent nitrosative stress in CLL cells may lead to hyperactive mitochondrial biogenesis. Here we report that primary CLL cells contained significantly more mitochondria than normal lymphocytes and that their mitochondrial mass was significantly related to endogenous NO levels. Expression of the mitochondrial biogenesis factors nuclear respiratory factor-1 and mitochondrial transcription factor A was elevated in most CLL specimens examined and appeared to be related to cellular NO levels. Treatment of B cells with exogenous NO caused a substantial increase in mitochondrial mass. In vitro sensitivity of CLL cells to fludarabine was highly related to mitochondrial mass in that cells with greater mitochondrial mass were less sensitive to the drug. Taken together, our results suggest that NO is a key mediator of mitochondrial biogenesis in CLL and that modulation of mitochondrial biogenesis by NO may alter cellular sensitivity to fludarabine.

Surrogate markers in antiangiogenesis clinical trials.

Novel antiangiogenic agents currently being developed may ultimately be more effective against solid tumours and less toxic than cytotoxic chemotherapy. As a result of the early clinical trials of angiogenesis inhibitors, investigators are beginning to appreciate the complexity of targeting angiogenesis and the realisation that developing clinically useful antiangiogenic therapy will be more challenging than originally thought. It is now apparent that new methods and surrogate markers to assess these agents' biological activity are crucial for their successful development. This review summarises the currently available clinical data on the development of surrogate markers of angiogenesis inhibitors.

p53 and Fas ligand are required for psoralen and UVA-induced apoptosis in mouse epidermal cells.

A combination of 8-methoxypsoralen (8-MOP) and ultraviolet-A (UVA) radiation (320-400 nm) (PUVA) is widely used in the treatment of psoriasis and other skin diseases. PUVA is highly effective in eliminating hyperproliferative cells in the epidermis, but its mechanism of action has not been fully elucidated. In this study, we used immortalized JB6 mouse epidermal cells, p53(-/-), and Fas ligand deficient (gld) mice to investigate the molecular mechanism by which PUVA induces cell death. The results indicate that PUVA treatment induces apoptosis in JB6 cells. In addition, PUVA treatment of JB6 cells results in p53 stabilization, phosphorylation, and nuclear localization as well as induction of p21(Waf/Cip1) and caspase-3 activity. In vivo studies reveal that PUVA treatment induces significantly less apoptosis in the epidermis of p53(-/-) mice compared to p53(+/+) mice. Furthermore, FasL-deficient (gld) mice are completely resistant to PUVA-induced apoptosis compared to wild-type mice. These results indicate that PUVA treatment induces apoptosis in mouse epidermal cells in vitro and in vivo and that p53 and Fas/Fas ligand interactions are required for this process, at least in vivo. This implies that similar mechanisms may be involved in the elimination of psoriatic keratinocytes from human skin following PUVA therapy.

Histone deacetylase inhibitor downregulation of bcl-xl gene expression leads to apoptotic cell death in mesothelioma.

It has been shown that mesothelioma expresses the antiapoptotic protein BCL-XL, but not BCL-2, rendering bcl-xl gene expression a potential therapeutic target. Sodium butyrate (NaB) is a histone deacetylase inhibitor capable of alteration of bcl-2 family protein expression in other tumor types. Mesothelioma cell lines (REN, I-45) were exposed to NaB, and viability (colorimetric assay) and apoptosis (TUNEL, Hoescht staining, flow cytometry) were evaluated. Effects on bcl-2 family protein, fas-fas ligand, and caspases were examined by Western blot analysis and functional assay. An RNase assay evaluated bcl-2 family messenger RNA (mRNA) expression. Overexpressing BCL-XL mesothelioma clones were created by plasmid transfer. Cells were sensitive to NaB at low IC(50) (REN, 0.3 mM; I-45, 1 mM) and demonstrated apoptosis (percentage of cells below G1 phase by flow cytometry [sub-G1]: REN, 38.5%; I-45, 30.9%). A significant decrease in BCL-XL protein expression was noted with BAK, BAX, and BCL-2 unchanged, and this was corroborated at the transcriptional level with selectively decreased bcl-xl mRNA production after sodium butyrate exposure. Fas expression and fas-fas ligand sensitivity were unchanged. Caspases demonstrated low-level activation. Stable overexpressing BCL-XL clones were proportionally resistant to the NaB effect. This study suggests that mesothelioma cells are sensitive to the induction of apoptosis related to the attenuation of antiapoptotic bcl-xl gene and protein expression. Additional study of the therapeutic benefit of targeting bcl-xl gene expression in mesothelioma is warranted.

In vivo intracellular signaling as a marker of antiangiogenic activity.

Alterations in endothelial cell (EC) signaling could serve as a marker of effective antiangiogenic therapy. We determined the effect of an antiangiogenic tyrosine kinase inhibitor, SU6668, on tumor EC signaling in liver metastases in mice. In vitro immunofluorescence verified that pretreatment of ECs with SU6668 before exposure to VEGF decreased in vitro phosphorylation of Erk and Akt. Using double-fluorescence immunohistochemistry, phosphorylated Erk and Akt were constitutively expressed in ECs in liver metastases in untreated mice, but SU6668 blocked activation of these signaling intermediates. Determining the activation status of the Erk and Akt signaling pathways in tumor ECs may serve as a surrogate marker for the effectiveness of antiangiogenic regimens.

Treatment with low-dose interferon-alpha restores the balance between matrix metalloproteinase-9 and E-cadherin expression in human transitional cell carcinoma of the bladder.

Tumor invasion and metastasis are regulated by the expression of genes such as E-cadherin, which regulates cell adhesion, and matrix metalloproteinase-9 (MMP-9), which alters the integrity of the extracellular matrix. Both up-regulation of MMP-9 and down-regulation of E-cadherin correlate with bladder cancer metastasis. The purpose of this study was first to determine whether an imbalance between MMP-9 and E-cadherin expression correlates with metastasis from human transitional cell carcinoma (TCC) of the bladder after therapy with neoadjuvant chemotherapy and radical cystectomy and then to determine whether treatment of human TCC xenografts growing in nude mice with interferon (IFN)-alpha would restore this balance, thereby limiting tumor invasion and metastasis. We used in situ hybridization to evaluate the expression of several metastasis-related genes, including MMP-9 and E-cadherin, in paraffin-embedded biopsy specimens from 55 patients with muscle-invasive TCC treated with neoadjuvant methotrexate, vinblastine, doxorubicin, and cisplatin chemotherapy and radical cystectomy. By multivariate analysis, an MMP-9:E-cadherin ratio of >1.8 was an independent prognostic factor for disease progression. In vitro incubation of an IFN-resistant, highly metastatic human TCC cell line, 253J B-V(R) with noncytostatic concentrations of IFN-alpha down-regulated the activity of MMP-9, up-regulated E-cadherin, and inhibited in vitro invasion. 253J B-V(R) cells were implanted into the bladders of athymic nude mice. Systemic therapy with IFN-alpha (10,000 units s.c. daily) decreased the expression of MMP-9, increased expression of E-cadherin, reduced tumor volume, and inhibited metastasis. The MMP-9:E-cadherin ratio was 4.5 in untreated controls and 1.1 after IFN-alpha treatment. Moreover, systemic low-dose daily IFN-alpha potentiated the efficacy of paclitaxel. These studies indicate that in addition to its antiproliferative and antiangiogenic effects, IFN-alpha limits tumor invasion by restoring the normal balance between MMP-9 and E-cadherin and enhances the activity of systemic chemotherapy.

BCL2 expression correlates with metastatic potential in pancreatic cancer cell lines.

BACKGROUND: Programmed cell death (termed apoptosis) regulates normal tissue homeostasis. Loss of local paracrine signals and intercellular adhesion molecules are potent inducers of apoptosis and thereby eliminate normal cells that may have escaped beyond the confines of the local organ environment. Dysregulation in the expression of the BCL2 gene family, the prototypic regulators of apoptosis, is a common occurrence in cancer and imparts resistance to standard triggers of apoptosis. Therefore, the authors sought to examine whether abnormal BCL2 gene family expression correlated with resistance to apoptosis and increased metastatic potential in pancreatic carcinoma. METHODS: The authors examined BCL2 expression and apoptotic sensitivity in three panels of human pancreatic cancer cell lines that possess varying metastatic potential. Stable transfectants were generated that overexpress BCL2. These transfectants were then analyzed for differences in metastasis formation in athymic mice. RESULTS: Among the isogenic panels of pancreatic cancer cell lines, BCL2 expression levels correlated with metastatic potential. Highly metastatic variants of each family of cell lines were more resistant to induction of apoptosis. Finally, using the BCL2 transfectant in a xenograft model, elevated BCL2 expression led to a higher incidence of metastases. CONCLUSIONS: The authors conclude that increased BCL2 expression correlates with apoptotic resistance and metastatic potential; dysregulation of BCL2 expression may be involved in the metastatic progression of pancreatic carcinoma.

Chemosensitization of pancreatic cancer by inhibition of the 26S proteasome.

BACKGROUND: Pancreatic cancer is extremely resistant to the induction of apoptosis by chemotherapies; agents that regulate sensitivity to apoptosis may lead to chemosensitization of pancreatic cancer. MATERIALS AND METHODS: MIA-PaCa-2 human pancreatic cancer cells were treated in vitro with the 26S-proteasome inhibitor PS-341. Levels of the apoptosis-regulating proteins (BCL-2, BAK, and BAX) were determined by Western blotting. The effect of PS-341 on BCL-2 gene transcription was examined using a BCL-2 promoter/luciferase reporter construct. The chemosensitizing effect of PS-341 was determined by measurement of the cytotoxic effect of gemcitabine in the presence of PS-341 (10-1000 nM) using the MTT assay. A corresponding in vivo experiment using tumor xenografts in athymic mice was also performed. RESULTS: PS-341 decreased BCL-2, without effect on BAX or BAK. The downregulation of BCL-2 by PS-341 appears to be transcriptionally mediated. PS-341 induced apoptosis at high does (1000 nM) and increased the cytotoxicity of gemcitabine at low doses (10-100 nM). Xenograft growth was inhibited 59% by gemcitabine; the addition of PS-341 increased growth inhibition to 75%. CONCLUSIONS: Inhibition of the 26S proteasome disrupts the cellular content of key regulators of cell cycle progression and apoptotic control leading to increased sensitivity to standard chemotherapeutic agents, such as gemcitabine, in pancreatic cancer. Combination therapy may lead to better response rates.

Calcium flux measurements in apoptosis.

Early studies in apoptosis implicated an increase in cytosolic Ca2+ as a direct mediator of DNA fragmentation. However, efforts to delineate targets for this increase in Ca2+ have been slow in evolving. Several previous studies have implicated ER Ca2+ pool depletion in the initiation of apoptosis. Our own preliminary studies confirm that many (but not all) apoptotic stimuli empty the ER store via a mechanism that is blocked by BCL-2 expression. Furthermore, ER pool depletion is not affected by broad spectrum caspase inhibitors, indicating that it occurs via a caspase-independent mechanism. Finally, our data demonstrate that ER pool depletion occurs prior to release of cytochrome c from mitochondria. Given previous work demonstrating close coordination of ER and mitochondrial Ca2+ levels, we speculate that ER-dependent changes in mitochondrial Ca2+ serve as important signals for cytochrome c release. Alternative mechanisms include activation of caspase-12 and/or the JNK pathway, both of which can be directly stimulated by depletion of the ER Ca2+ pool. Although substantial improvements in intracellular Ca2+ imaging have emerged, compelling answers to many of the present questions related to the role of Ca2+ in apoptosis await future technical improvements. The development of organelle-specific, recombinant Ca2+ probes (targeted aequorins and cameleons) certainly should facilitate some of this work, although the target cell of interest must be amenable to molecular manipulation (transfection), which precludes straightforward analysis of primary cells. Pharmacological tools (i.e., thapsigargin and DBHQ) can provide conclusive data on ER pool status without requiring an overly sophisticated image analysis system. However, confocal microscopy allows for the effective analysis of Ca2+ pools as long as dye localization is homogeneous and properly controlled. However, current techniques should be considered semiquantitative at best and will remain so until specific organelle-targeted fluorescent dyes are developed and widely available.

Endothelial cell apoptosis is inhibited by a soluble factor secreted by human colon cancer cells.

In contrast to normal tissues, tumors are exposed to adverse environmental conditions, such as hypoxia and acidity. Despite this caustic environment, tumor cells and supporting vascular structures survive the latter, providing nutrients and oxygen to facilitate tumor growth. Therefore, we hypothesized that cancer cells express factors that protect endothelial cells from apoptosis. Human umbilical vein endothelial cells were grown in serum-free medium or in medium conditioned by either human colon cancer cells or non-malignant cells. Endothelial cells grown in medium conditioned by colon cancer cells demonstrated a decrease in apoptosis, whereas endothelial cells grown in medium conditioned by non-malignant cells did not. Erk-1/2 phosphorylation increased when endothelial cells were incubated in medium conditioned by colon cancer cells but not when cells were incubated in medium conditioned by non-malignant cells. Protein fractions from medium conditioned by colon cancer cells that were < 3 kDa protected endothelial cells from apoptosis and activated Erk-1/2. Heat-inactivated conditioned media did not protect endothelial cells from apoptosis and did not activate Erk-1/2. Human colon cancer cells secrete a soluble factor or factors that inhibit endothelial cell apoptosis. This factor is likely to be a protein or protein fragment because it loses its activity after heat inactivation. These studies support the hypothesis that tumor cells can alter the phenotype of endothelial cells.

Tyrosine kinase inhibition of multiple angiogenic growth factor receptors improves survival in mice bearing colon cancer liver metastases by inhibition of endothelial cell survival mechanisms.

Redundant mechanisms mediate colon cancer angiogenesis. Targeting multiple angiogenic factors simultaneously may improve survival of mice with colon cancer metastases. BALB/c mice underwent splenic injection with CT-26 colon cancer cells to generate liver metastases and received administration of either vehicle alone or a tyrosine kinase inhibitor for vascular endothelial growth factor, basic fibroblast growth factor, and platelet-derived growth factor receptors (SU6668). Mice were sacrificed when they became moribund as determined by a blinded observer. In a parallel experiment, groups of mice were sacrificed at earlier time points to better define the kinetics of the effect of SU6668 on angiogenic parameters over time. SU6668 increased median survival by 58% (P < 0.001) and led to a progressive increase in tumor cell and endothelial cell apoptosis that increased over time. In addition, pericyte vessel coverage and tumor vascularity were significantly decreased in mice treated with SU6668. Based on current knowledge of endothelial cell survival, these data suggest that SU6668 may prevent tumor endothelial cell survival directly (vascular endothelial growth factor) and indirectly (pericyte coverage) by affecting endothelial cell survival mechanisms.

Spontaneous regression of cutaneous melanoma in sinclair swine is associated with defective telomerase activity and extensive telomere erosion.

Recently we proposed the hypothesis that extensive telomeric association of chromosomes is an early manifestation of cell death and asked whether there are extensive telomeric associations present in metaphases of the spontaneously regressing Sinclair swine cutaneous melanoma (SSCM). Our results indicate that early passage SSCMs, in the accelerated growth phase, do not show telomeric associations but do have numerical and other specific structural abnormalities. However, the same melanoma cell lines at late passages or melanomas obtained from middle- and old-aged Sinclair swine show extensive telomeric associations in the form of dicentric, multicentric, and ring configurations. Such abnormal structures are present mostly in metaphases that are hyperploids. Increasing frequencies of apoptotic bodies were also observed in higher passage tumor cell lines obtained from younger animals or in melanomas obtained from older animals. The polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) assay shows no detectable telomerase activity in any of these regressing swine melanoma cell lines, neither in normal swine skin fibroblasts nor in nevi. However, the fetal swine (i.e., non-regressing) melanoma cells show telomerase activity. Fluorescence in situ hybridization (FISH) results using the commercially available human telomeric repeat DNA probe indicate a reduction of telomeric signals in metaphase and interphase cells of regressing melanomas. From these observations we conclude that spontaneous regression of SSCM is associated with the loss of telomerase activity and a reduction of telomeric repeats that results in the formation of multicentric and ring configurations. Such abnormal chromosome configurations are lost, following the breakage-fusion-bridge-cycles, and result in extensive DNA fragmentation, as shown by laddering experiments, and, finally, cell death.

Nitric oxide-dependent activation of p53 suppresses bleomycin-induced apoptosis in the lung.

Chronic inflammation leading to pulmonary fibrosis develops in response to environmental pollutants, radiotherapy, or certain cancer chemotherapeutic agents. We speculated that lung injury might be mediated by p53, a proapoptotic transcription factor widely implicated in the response of cells to DNA damage. Intratracheal administration of bleomycin led to caspase-mediated DNA fragmentation characteristic of apoptosis. The effects of bleomycin were associated with translocation of p53 from the cytosol to the nucleus only in alveolar macrophages that had been exposed to the drug in vivo, suggesting that the lung microenvironment regulated p53 activation. Experiments with a thiol antioxidant (N-acetylcysteine) in vivo and nitric oxide (NO) donors in vitro confirmed that reactive oxygen species were required for p53 activation. A specific role for NO was demonstrated in experiments with inducible nitric oxide synthase (iNOS)(-/)- macrophages, which failed to demonstrate nuclear p53 localization after in vivo bleomycin exposure. Strikingly, rates of bleomycin-induced apoptosis were at least twofold higher in p53(-/)- C57BL/6 mice compared with heterozygous or wild-type littermates. Similarly, levels of apoptosis were also twofold higher in the lungs of iNOS(-/)- mice than were observed in wild-type controls. Consistent with a role for apoptosis in chronic lung injury, levels of bleomycin-induced inflammation were substantially higher in iNOS(-/)- and p53(-/)- mice compared with wild-type controls. Together, our results demonstrate that iNOS and p53 mediate a novel apoptosis-suppressing pathway in the lung.

Expression of vascular endothelial growth factor is necessary but not sufficient for production and growth of brain metastasis.

We investigated the molecular mechanisms of angiogenesis in experimental brain metastasis. Cells from six different human cancer cell lines (proven to produce visceral metastasis) were injected into the internal carotid artery of nude mice. Colon carcinoma (KM12SM) and lung adenocarcinoma (PC14PE6 and PC14Br) cells produced large, fast-growing parenchymal brain metastases, whereas lung squamous cell carcinoma (H226), renal cell carcinoma (SN12PM6), and melanoma (TXM13) cells produced only a few slow-growing brain metastases. Rapidly progressing brain metastases contained many enlarged blood vessels. The expression of VEGF mRNA and protein by the tumor cells directly correlated with angiogenesis and growth of brain metastasis. Causal evidence for the essential role of VEGF in this process was provided by transfecting PC14PE6 and KM12SM cells with antisense-VEGF165 gene, which significantly decreased the incidence of brain metastasis. In contrast, transfection of H226 human lung squamous carcinoma cells with sense-VEGF121 or sense-VEGF165 neither enhanced nor inhibited formation of brain metastases. Collectively, the results indicate that VEGF expression is necessary but not sufficient for the production of brain metastasis and that the inhibition of VEGF represents an important therapeutic target.