Does the extent of neck surgery based on preoperative calcitonin level influence survival in medullary thyroid carcinoma: a retrospective tertiary centre experience.

INTRODUCTION: Medullary thyroid carcinoma (MTC) is a rare thyroid cancer arising from the calcitonin-secreting parafollicular cells. Previous studies suggested a preoperative calcitonin level >200ng/l is an indication for prophylactic lateral neck dissection (LND) to remove micrometastases and improve survival outcomes. METHODS: This retrospective single-centre study assessed the efficacy of preoperative calcitonin levels as a marker for determining need for prophylactic LND in MTC. Data were obtained on demographics, preoperative calcitonin levels, size and laterality of index tumour, type of neck dissection (central neck dissection (CND), LND), nodes removed, levels with involved nodes, number of nodes histologically involved, mortality, adjuvant therapy and locoregional recurrence. RESULTS: A total of 33 patients were identified from St George's University Hospitals NHS Foundation Trust between 1 January 2001 and 19 August 2021; 8 were excluded for data quality issues. Of the 18 classified with a high preoperative calcitonin level (>200ng/l), 10 (56%) had a LND and 8 (44%) had a CND. In the low-calcitonin group, three (43%) patients had a CND only and four (57%) had a LND. There was no difference in absolute or disease-free survival between the low and high groups (p=0.960, p=0.817), or between those who had a CND and LND in the high-calcitonin group (p=0.607, hazard ratio (HR) 0.55; p=0.129, HR 8.78). CONCLUSION: There was no statistically significant difference in outcomes between high and low calcitonin groups. A selective approach to performing LND in MTC patients based on clinical and imaging findings suggesting disease presence in the lateral neck should be explored further.

Chemoprevention gene therapy (CGT) of pancreatic cancer using perillyl alcohol and a novel chimeric serotype cancer terminator virus.

Conditionally replication competent adenoviruses (Ads) that selectively replicate in cancer cells and simultaneously express a therapeutic cytokine, such as melanoma differentiation associated gene- 7/Interleukin-24 (mda-7/IL-24), a Cancer Terminator Virus (CTV-M7), hold potential for treating human cancers. To enhance the efficacy of the CTV-M7, we generated a chimeric Ad.5 and Ad.3 modified fiber bipartite CTV (Ad.5/3-CTV-M7) that can infect tumor cells in a Coxsackie Adenovirus receptor (CAR) independent manner, while retaining high infectivity in cancer cells containing high CAR. Although mda-7/IL-24 displays broad-spectrum anticancer properties, pancreatic ductal adenocarcinoma (PDAC) cells display an intrinsic resistance to mda-7/IL-24-mediated killing due to an mda-7/IL-24 mRNA translational block. However, using a chemoprevention gene therapy (CGT) approach with perillyl alcohol (POH) and a replication incompetent Ad to deliver mda-7/IL-24 (Ad.mda-7) there is enhanced conversion of mda-7/IL-24 mRNA into protein resulting in pancreatic cancer cell death in vitro and in vivo in nude mice containing human PDAC xenografts. This combination synergistically induces mda-7/IL-24-mediated cancer-specific apoptosis by inhibiting anti-apoptotic Bcl-xL and Bcl-2 protein expression and inducing an endoplasmic reticulum (ER) stress response through induction of BiP/GRP-78, which is most evident in chimeric-modified non-replicating Ad.5/3- mda-7- and CTV-M7-infected PDAC cells. Moreover, Ad.5/3-CTV-M7 in combination with POH sensitizes therapy-resistant MIA PaCa-2 cell lines over-expressing either Bcl-2 or Bcl-xL to mda-7/IL-24-mediated apoptosis. Ad.5/3-CTV-M7 plus POH also exerts a significant antitumor 'bystander' effect in vivo suppressing both primary and distant site tumor growth, confirming therapeutic utility of Ad.5/3-CTV-M7 plus POH in PDAC treatment, where all other current treatment strategies in clinical settings show minimal efficacy.

Sphingosine-1-phosphate phosphohydrolase-1 regulates ER stress-induced autophagy.

The sphingolipid metabolites ceramide and sphingosine-1-phosphate (S1P) have recently been implicated in autophagy. In this study, we report that depletion of sphingosine-1-phosphate phosphohydrolase-1 (SPP1), an endoplasmic reticulum (ER)-resident enzyme that specifically dephosphorylates S1P, induced autophagy. Although the mammalian target of rapamycin and class III phosphoinositide 3-kinase/Beclin-1 pathways were not involved and this autophagy was p53 independent, C/EBP homologous protein, BiP, and phospho-eucaryotic translation initiation factor-2α, and cleavage of procaspases 2 and 4, downstream targets of ER stress, were increased after SPP1 depletion. Autophagy was suppressed by depletion of protein kinase regulated by RNA-like ER kinase (PERK), inositol-requiring transmembrane kinase/endonuclease-1α, or activating transcription factor 6, three sensors of the unfolded protein response (UPR) to ER stress. Autophagy triggered by downregulation of SPP1 did not lead to apoptosis but rather stimulated, in a PERK dependent manner, the survival signal Akt, whose inhibition then sensitized cells to apoptosis. Although depletion of SPP1 increased intracellular levels of S1P and its secretion, activation of cell surface S1P receptors did not induce autophagy. Nevertheless, increases in intracellular pools of S1P, but not dihydro-S1P, induced autophagy and ER stress. Thus, SPP1, by regulating intracellular S1P homeostasis, can control the UPR and ER stress-induced autophagy.

Prevalent vertebral fractures among children initiating glucocorticoid therapy for the treatment of rheumatic disorders.

OBJECTIVE: Vertebral fractures are an under-recognized problem in children with inflammatory disorders. We studied spine health among 134 children (87 girls) with rheumatic conditions (median age 10 years) within 30 days of initiating glucocorticoid therapy. METHODS: Children were categorized as follows: juvenile dermatomyositis (n = 30), juvenile idiopathic arthritis (n = 28), systemic lupus erythematosus and related conditions (n = 26), systemic arthritis (n = 22), systemic vasculitis (n = 16), and other conditions (n = 12). Thoracolumbar spine radiograph and dual x-ray absorptiometry for lumbar spine (L-spine) areal bone mineral density (BMD) were performed within 30 days of glucocorticoid initiation. Genant semiquantitative grading was used for vertebral morphometry. Second metacarpal morphometry was carried out on a hand radiograph. Clinical factors including disease and physical activity, calcium and vitamin D intake, cumulative glucocorticoid dose, underlying diagnosis, L-spine BMD Z score, and back pain were analyzed for association with vertebral fracture. RESULTS: Thirteen vertebral fractures were noted in 9 children (7%). Of these, 6 patients had a single vertebral fracture and 3 had 2-3 fractures. Fractures were clustered in the mid-thoracic region (69%). Three vertebral fractures (23%) were moderate (grade 2); the others were mild (grade 1). For the entire cohort, mean +/- SD L-spine BMD Z score was significantly different from zero (-0.55 +/- 1.2, P < 0.001) despite a mean height Z score that was similar to the healthy average (0.02 +/- 1.0, P = 0.825). Back pain was highly associated with increased odds for fracture (odds ratio 10.6 [95% confidence interval 2.1-53.8], P = 0.004). CONCLUSION: In pediatric rheumatic conditions, vertebral fractures can be present prior to prolonged glucocorticoid exposure.

Enhanced delivery of mda-7/IL-24 using a serotype chimeric adenovirus (Ad.5/3) improves therapeutic efficacy in low CAR prostate cancer cells.

Gene therapy is being examined as a potential strategy for treating prostate cancer. Serotype 5 adenovirus (Ad.5) is routinely used as a vector for transgene delivery. However, the infectivity of Ad.5 is dependent on Coxsackie-adenovirus receptors (CARs); many tumor types show a reduction in this receptor in vivo, thereby limiting therapeutic gene transduction. Serotype chimerism is one approach to circumvent CAR deficiency; this strategy is used to generate an Ad.5/3-recombinant Ad that infects cancer cells through Ad.3 receptors in a CAR-independent manner. In this report, the enhanced transgene delivery and efficacy of Ad.5/3-recombinant virus was evaluated using an effective wide-spectrum anticancer therapeutic melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24). Our data show that in low CAR human prostate cancer cells (PC-3), a recombinant Ad.5/3 virus delivering mda-7/IL-24 (Ad.5/3-mda-7) is more efficacious than an Ad.5 virus encoding mda-7/IL-24 (Ad.5-mda-7) in infecting tumor cells, expressing MDA-7/IL-24 protein, inducing cancer-specific apoptosis, inhibiting in vivo tumor growth and exerting an antitumor 'bystander' effect in a nude mouse xenograft model. Considering the fact that Ad.5-mda-7 has shown significant objective responses in a phase I clinical trial for solid tumors, Ad.5/3-mda-7 is predicted to exert enhanced therapeutic benefit in patients with prostate cancer.

Characterization of infectivity of knob-modified adenoviral vectors in glioma.

Malignant glioma continues to be a major target for gene therapy and virotherapy due to its aggressive growth and the current lack of effective treatment. However, these approaches have been hampered by inefficient infection of glioma cells by viral vectors,particularly vectors derived from serotype 5 adenoviruses (Ad5). This results from limited cell surface expression of the primary adenovirus receptor, coxsackie-adenovirus-receptor (CAR), on tumor cells. To circumvent this problem, Ad fiber pseudotyping,the genetic replacement of either the entire fiber or fiber knob domain with its structural counterpart from another human Ad serotype that recognizes a cellular receptor other than CAR, has been shown to enhance Ad infectivity in a variety of tumor types,including human glioma. Here, we have extended the paradigm of genetic pseudotyping to include fiber domains from non-human or"xenotype" Ads for infectivity enhancement of human glioma cell populations. In this study, we evaluated the gene transfer efficiency of a panel of Ad vectors which express one of five different "xenotype"fiber knob domains, including those derived from murine,ovine, porcine and canine species, in both human glioma cell lines as well as primary glioma tumor cells from patients. Adenovirus vectors displaying either canine Ad or porcine Ad fiber elements had the highest gene transfer to both glioma cell lines and primary tumor cells. The correlation between the viral infectivity of modified adenovirus vectors and expression of human CAR and CD46(an adenovirus type B receptor) on the surfaces of tumor cells was also analyzed. Taken together, human adenovirus vectors modified with "xenotype" fiber elements could be excellent candidates to target human glioma.

A cancer terminator virus eradicates both primary and distant human melanomas.

The prognosis and response to conventional therapies of malignant melanoma inversely correlate with disease progression. With increasing thickness, melanomas acquire metastatic potential and become inherently resistant to radiotherapy and chemotherapy. These harsh realities mandate the design of improved therapeutic modalities, especially those targeting metastases. To develop an approach to effectively treat this aggressive disease, we constructed a conditionally replication-competent adenovirus in which expression of the adenoviral E1A gene, necessary for replication, is driven by the cancer-specific promoter of progression-elevated gene-3 (PEG-3) and which simultaneously expresses mda-7/IL-24 in the E3 region of the adenovirus (Ad.PEG-E1A-mda-7), a cancer terminator virus (CTV). This CTV produces large quantities of MDA-7/IL-24 protein as a function of adenovirus replication uniquely in cancer cells. Infection of Ad.PEG-E1A-mda-7 (CTV) in normal human immortal melanocytes and human melanoma cells demonstrates cancer cell-selective adenoviral replication, mda-7/IL-24 expression, growth inhibition and apoptosis induction. Injecting Ad.PEG-E1A-mda-7 CTV into xenografts derived from MeWo human metastatic melanoma cells in athymic nude mice completely eliminated not only primary treated tumors but also distant non-treated tumors (established in the opposite flank), thereby implementing a cure. These provocative findings advocate potential therapeutic applications of this novel virus for treating patients with advanced melanomas with metastases.

Activation of Ras/Raf protects cells from melanoma differentiation-associated gene-5-induced apoptosis.

Melanoma differentiation-associated gene-5 (mda-5) was the first molecule identified in nature whose encoded protein embodied the unique structural combination of an N-terminal caspase recruitment domain and a C-terminal DExD/H RNA helicase domain. As suggested by its structure, cumulative evidences documented that ectopic expression of mda-5 leads to growth inhibition and/or apoptosis in various cell lines. However, the signaling pathways involved in mda-5-mediated killing have not been elucidated. In this study, we utilized either genetically modified cloned rat embryo fibroblast cells overexpressing different functionally and structurally distinct oncogenes or human pancreatic and colorectal carcinoma cells containing mutant active ras to resolve the role of the Ras/Raf signaling pathway in mda-5-mediated growth inhibition/apoptosis induction. Rodent and human tumor cells containing constitutively activated Raf/Raf/MEK/ERK pathways were resistant to mda-5-induced killing and this protection was antagonized by intervening in this signal transduction cascade either by directly inhibiting ras activity using an antisense strategy or by targeting ras-downstream factors, such as MEK1/2, with the pharmacological inhibitor PD98059. The present findings provide a further example of potential cross-talk between growth-inhibitory and growth-promoting pathways in which the ultimate balance of these factors defines cellular homeostasis, leading to survival or induction of programmed cell death.

Ionizing radiation enhances therapeutic activity of mda-7/IL-24: overcoming radiation- and mda-7/IL-24-resistance in prostate cancer cells overexpressing the antiapoptotic proteins bcl-xL or bcl-2.

Subtraction hybridization applied to terminally differentiating human melanoma cells identified mda-7/IL-24, a cytokine belonging to the IL-10 gene superfamily. Adenoviral-mediated delivery of mda-7/IL-24 (Ad.mda-7) provokes apoptosis selectively in a wide spectrum of cancers in vitro in cell culture, in vivo in human tumor xenograft animal models and in patients with advanced carcinomas and melanomas. In human prostate cancer cells, a role for mitochondrial dysfunction and induction of reactive oxygen species in the apoptotic process has been established. Ectopic overexpression of bcl-xL and bcl-2 prevents these changes including apoptosis induction in prostate tumor cells by Ad.mda-7. We now document that this resistance to apoptosis can be reversed by treating bcl-2 family overexpressing prostate tumor cells with ionizing radiation in combination with Ad.mda-7 or purified GST-MDA-7 protein. Additionally, radiation augments apoptosis induction by mda-7/IL-24 in parental and neomycin-resistant prostate tumor cells. Radiosensitization to mda-7/IL-24 is dependent on JNK signaling, as treatment with the JNK 1/2/3 inhibitor SP600125 abolishes this effect. Considering that elevated expression of bcl-xL and bcl-2 are frequent events in prostate cancer development and progression, the present studies support the use of ionizing radiation in combination with mda-7/IL-24 as a means of augmenting the therapeutic benefit of this gene in prostate cancer, particularly in the context of tumors displaying resistance to radiation therapy owing to bcl-2 family member overexpression.

Nitric oxide inhibition and the impact on renal nerve-mediated antinatriuresis and antidiuresis in the anaesthetized rat.

The contribution of nitric oxide (NO) to the antinatriuresis and antidiuresis caused by low-level electrical stimulation of the renal sympathetic nerves (RNS) was investigated in rats anaesthetized with chloralose-urethane. Groups of rats, n= 6, were given i.v. infusions of vehicle, l-NAME (10 microg kg(-1) min(-1)), 1400W (20 microg kg(-1) min(-1)), or S-methyl-thiocitrulline (SMTC) (20 microg kg(-1) min(-1)) to inhibit NO synthesis non-selectively or selectively to block the inducible or neuronal NOS isoforms (iNOS and nNOS, respectively). Following baseline measurements of blood pressure (BP), renal blood flow (RBF), glomerular filtration rate (GFR), urine flow (UV) and sodium excretion (U(Na)V), RNS was performed at 15 V, 2 ms duration with a frequency between 0.5 and 1.0 Hz. RNS did not cause measurable changes in BP, RBF or GFR in any of the groups. In untreated rats, RNS decreased UV and U(Na)V by 40-50% (both P < 0.01), but these excretory responses were prevented in l-NAME-treated rats. In the presence of 1400W i.v., RNS caused reversible reductions in both UV and U(Na)V of 40-50% (both P < 0.01), while in SMTC-treated rats, RNS caused an inconsistent fall in UV, but a significant reduction (P < 0.05) in U(Na)V of 21%. These data demonstrated that the renal nerve-mediated antinatriuresis and antidiuresis was dependent on the presence of NO, generated in part by nNOS. The findings suggest that NO importantly modulates the neural control of fluid reabsorption; the control may be facilitatory at a presynaptic level but inhibitory on tubular reabsorptive processes.

Synergistic interactions between MEK1/2 and histone deacetylase inhibitors in BCR/ABL+ human leukemia cells.

Interactions between the histone deacetylase inhibitor SAHA and the pharmacologic MEK1/2 inhibitor PD184352 were examined in Bcr/Abl+ human leukemia cells. Coadministration of minimally toxic concentrations of SAHA (or sodium butyrate) and PD184352 (or U0126) resulted in a synergistic increase in mitochondrial damage, caspase activation, and apoptosis in K562 and LAMA 84 cells. Similar interactions were observed in CD34+ cells from two patients with CML and in imatinib mesylate-resistant K562 cells but not in normal human CD34+ bone marrow cells. These events were associated with a marked increase in ROS generation, inactivation of ERK and Akt, downregulation of p21CIP1, Bcr/Abl, and cyclin D1, and activation of JNK. Of these events, ROS generation, ERK inactivation, and cytochrome c/AIF release were largely caspase-independent, whereas the other phenomena displayed varying degrees of caspase-dependence. Using pharmacologic and genetic approaches, generation of ROS, p21CIP1 downregulation, and inactivation of Akt and MEK were found to play significant functional roles in SAHA/PD184352-mediated lethality, whereas JNK activation and Raf-1 downregulation were determined to represent secondary events. These findings indicate that interruption of the MEK/ERK pathway substantially lowers the threshold for HDAC inhibitor-mediated oxidative injury, mitochondrial dysfunction, and apoptosis, suggesting that this approach warrants further examination in Bcr/Abl+-related malignancies.

Synergistic induction of apoptosis in human myeloid leukemia cells by phorbol 12-myristate 13-acetate and flavopiridol proceeds via activation of both the intrinsic and tumor necrosis factor-mediated extrinsic cell death pathways.

Previous studies have shown that coexposure to marginally toxic concentrations of phorbol 12-myristate 13-acetate (PMA; 10 nM) and the cyclin-dependent kinase inhibitor flavopiridol (FP; 100-200 nM) synergistically induces apoptosis in human myeloid leukemia cells U937 and HL-60 (i.e., >50% apoptotic at 24 h). Attempts have now been made to characterize the cell death pathway(s) involved in this phenomenon. In contrast to cytochrome c release and caspase-3 activation, which occur within 2.5 h of PMA/FP coexposure, caspase-8 activation and Bid cleavage appeared as later events. Such findings implicate the mitochondria-dependent pathway in the initial induction of apoptosis by PMA/FP. However, U937 cells ectopically expressing CrmA, dominant-negative caspase-8, or dominant-negative Fas-associated death domain that were highly resistant to tumor necrosis factor (TNF)/cycloheximide-induced lethality displayed significant, albeit incomplete, resistance to PMA/FP-induced apoptosis after 24 h. Furthermore, coadministration of TNF soluble receptor significantly attenuated PMA/FP-induced apoptosis in U937 (p < 0.02) and HL-60 (p < 0.03) cells at 24 h. PMA/FP coadministration also triggered substantial increases in TNFalpha mRNA and protein secretion compared with the effects of PMA administered alone. The protein kinase C (PKC) inhibitor bisindolylmaleimide (1 microM) completely blocked PMA/FP-induced TNFalpha secretion in U937 cells and attenuated apoptosis. Taken together, these results suggest that coadministration of PMA with FP in myeloid leukemia cells initially triggers mitochondrial damage, an event followed by the PKC-dependent induction and release of TNFalpha, supporting a model in which the synergistic induction of leukemic cell apoptosis by this drug combination proceeds via both mitochondrial- and TNF receptor-related apoptotic pathways.

Autocrine loops with positive feedback enable context-dependent cell signaling.

We describe a mechanism for context-dependent cell signaling mediated by autocrine loops with positive feedback. We demonstrate that the composition of the extracellular medium can critically influence the intracellular signaling dynamics induced by extracellular stimuli. Specifically, in the epidermal growth factor receptor (EGFR) system, amplitude and duration of mitogen-activated protein kinase (MAPK) activation are modulated by the positive-feedback loop formed by the EGFR, the Ras-MAPK signaling pathway, and a ligand-releasing protease. The signaling response to a transient input is short-lived when most of the released ligand is lost to the cellular microenvironment by diffusion and/or interaction with an extracellular ligand-binding component. In contrast, the response is prolonged or persistent in a cell that is efficient in recapturing the endogenous ligand. To study functional capabilities of autocrine loops, we have developed a mathematical model that accounts for ligand release, transport, binding, and intracellular signaling. We find that context-dependent signaling arises as a result of dynamic interaction between the parts of an autocrine loop. Using the model, we can directly interpret experimental observations on context-dependent responses of autocrine cells to ionizing radiation. In human carcinoma cells, MAPK signaling patterns induced by a short pulse of ionizing radiation can be transient or sustained, depending on cell type and composition of the extracellular medium. On the basis of our model, we propose that autocrine loops in this, and potentially other, growth factor and cytokine systems may serve as modules for context-dependent cell signaling.

Differentiation therapy of human cancer: basic science and clinical applications.

Current cancer therapies are highly toxic and often nonspecific. A potentially less toxic approach to treating this prevalent disease employs agents that modify cancer cell differentiation, termed 'differentiation therapy.' This approach is based on the tacit assumption that many neoplastic cell types exhibit reversible defects in differentiation, which upon appropriate treatment, results in tumor reprogramming and a concomitant loss in proliferative capacity and induction of terminal differentiation or apoptosis (programmed cell death). Laboratory studies that focus on elucidating mechanisms of action are demonstrating the effectiveness of 'differentiation therapy,' which is now beginning to show translational promise in the clinical setting.

MAPK dependence of DNA damage repair: ionizing radiation and the induction of expression of the DNA repair genes XRCC1 and ERCC1 in DU145 human prostate carcinoma cells in a MEK1/2 dependent fashion.

PURPOSE: To examine the role of mitogen-activated protein kinase (MAPK) signalling on the induction by ionizing radiation of the nucleotide excision repair gene (ERCC1), the X-ray cross-complementing group 1 protein (XRCC1) and the repair of radiation-induced DNA damage. MATERIALS AND METHODS: The expression of ERCC1 and XRCC1 was examined in DU145 human prostate cancer cells following exposure to ionizing radiation. We characterized the MAPK dependence of this expression through RT-PCR analysis, Western analysis, transcription inhibition and measurement of the activation of each promoter. Pre-exposure with the specific MEK1/2 inhibitor PD980059 (10 microM) was used to blunt radiation induction of MAPK without suppressing basal levels of MAPK activity. In addition, we examined the MAPK dependence of DNA damage repair by measuring radiation-induced micronucleus formation and the removal of and nicking activity associated with AP sites. RESULTS: Irradiation caused a time-dependent, MAPK-dependent increase in the protein levels of both ERCC1 and XRCC1. For each gene product, the protein level increase followed an increase in mRNA, which also was MAPK-dependent. Radiation also enhanced the activities of the ERCC1 and XRCC1 promoters in an MAPK-dependent fashion. Inhibition of transcription by DRB abolished the radiation-induced increase of ERCC1 and XRCC1 proteins. Inhibition of radiation-induced MAPK also diminished the ability of DU145 cells to remove AP sites and increased the number of cells displaying micronuclei following radiation exposure. CONCLUSIONS: These findings demonstrate a role for radiation-induced MAPK signalling in the regulation of DNA repair enzyme levels and DNA repair. Radiation-induced protein expression of ERCC1 and XRCC1 appears to require de novo transcription. These data suggest a significant role for MAPK signalling in the early response to DNA damage caused by ionizing radiation.

Hepatitis B virus X protein increases expression of p21(Cip-1/WAF1/MDA6) and p27(Kip-1) in primary mouse hepatocytes, leading to reduced cell cycle progression.

Previously, we have linked prolonged intense mitogen-activated protein kinase (MAP kinase; MAPK) signaling in hepatocytes to increased expression of p21(Cip-1/WAF1/MDA6) (p21) and p16(INK4a) (p16), that leads to a p21-dependent growth arrest. In this study, we investigated the impact of hepatitis B virus X protein (pX) expression on MAPK-modulated cell cycle progression in primary mouse hepatocytes. In hepatocytes, expression of pX enhanced protein levels of p21 and p27, but not of p16. The elevated levels of p21 and p27 correlated with reduced DNA synthesis in wild-type (+/+) hepatocytes and with a weak stimulation of DNA synthesis in p21 null (-/-) cells. Antisense p27 messenger RNA (mRNA) (p27as) increased DNA synthesis in +/+ and p21 -/- cells, and pX blunted this effect in +/+ cells. In p21 -/- cells, however, p27as permitted pX to further stimulate DNA synthesis. These data argue that a reduced ability to enhance expression of both p21 and p27 is required to fully reveal the growth-potentiating properties of pX. This finding also implies that depending on the functional status of the p21 and p27 genes, expression of pX can have 2 very different effects on hepatocyte proliferation. Prolonged intense MAPK signaling reduced DNA synthesis in +/+ cells and enhanced DNA synthesis in p21 -/- cells. The enhancement of DNA synthesis in p21 -/- cells was blocked by pX, and the effect of pX was abrogated by p27as. Furthermore in p21 -/- cells, overexpression of p16 blocked MAPK-stimulated DNA synthesis, and this effect was partially reversed by p27as. These data argue that p27 can also cooperatively interact with p16 to inhibit DNA synthesis in hepatocytes. Collectively, our findings show that reduced expression of p16, p21, and p27, which can occur during hepatocellular carcinoma, enhances the ability of MAPK signaling and pX to cause proliferation in hepatocytes. Thus loss of cyclin kinase inhibitor function may play an important role in the process of tumor progression after chronic hepatitis B virus infection.

Deoxycholic acid (DCA) causes ligand-independent activation of epidermal growth factor receptor (EGFR) and FAS receptor in primary hepatocytes: inhibition of EGFR/mitogen-activated protein kinase-signaling module enhances DCA-induced apoptosis.

Previous studies have argued that enhanced activity of the epidermal growth factor receptor (EGFR) and the mitogen-activated protein kinase (MAPK) pathway can promote tumor cell survival in response to cytotoxic insults. In this study, we examined the impact of MAPK signaling on the survival of primary hepatocytes exposed to low concentrations of deoxycholic acid (DCA, 50 microM). Treatment of hepatocytes with DCA caused MAPK activation, which was dependent upon ligand independent activation of EGFR, and downstream signaling through Ras and PI(3) kinase. Neither inhibition of MAPK signaling alone by MEK1/2 inhibitors, nor exposure to DCA alone, enhanced basal hepatocyte apoptosis, whereas inhibition of DCA-induced MAPK activation caused approximately 25% apoptosis within 6 h. Similar data were also obtained when either dominant negative EGFR-CD533 or dominant negative Ras N17 were used to block MAPK activation. DCA-induced apoptosis correlated with sequential cleavage of procaspase 8, BID, procaspase 9, and procaspase 3. Inhibition of MAPK potentiated bile acid-induced apoptosis in hepatocytes with mutant FAS-ligand, but did not enhance in hepatocytes that were null for FAS receptor expression. These data argues that DCA is causing ligand independent activation of the FAS receptor to stimulate an apoptotic response, which is counteracted by enhanced ligand-independent EGFR/MAPK signaling. In agreement with FAS-mediated cell killing, inhibition of caspase function with the use of dominant negative Fas-associated protein with death domain, a caspase 8 inhibitor (Ile-Glu-Thr-Asp-p-nitroanilide [IETD]) or dominant negative procaspase 8 blocked the potentiation of bile acid-induced apoptosis. Inhibition of bile acid-induced MAPK signaling enhanced the cleavage of BID and release of cytochrome c from mitochondria, which were all blocked by IETD. Despite activation of caspase 8, expression of dominant negative procaspase 9 blocked procaspase 3 cleavage and the potentiation of DCA-induced apoptosis. Treatment of hepatocytes with DCA transiently increased expression of the caspase 8 inhibitor proteins c-FLIP-(S) and c-FLIP-(L) that were reduced by inhibition of MAPK or PI(3) kinase. Constitutive overexpression of c-FLIP-(s) abolished the potentiation of bile acid-induced apoptosis. Collectively, our data argue that loss of DCA-induced EGFR/Ras/MAPK pathway function potentiates DCA-stimulated FAS-induced hepatocyte cell death via a reduction in the expression of c-FLIP isoforms.

MEK1/2 inhibitors promote Ara-C-induced apoptosis but not loss of Deltapsi(m) in HL-60 cells.

The effects of pharmacologic MEK1/2 inhibitors on ara-C-mediated mitochondrial injury, caspase activation, and apoptosis have been examined in HL-60 leukemic cells. Coadministration of subtoxic concentrations of the MEK1/2 inhibitors U0126 (20 microM), PD98059 (40 microM), or PD184352 (10 microM) with 10-100 microM ara-C (6 h) potentiated apoptosis (i.e., by approx twofold), and pro-caspase 3, pro-caspase 8, Bid, and PARP cleavage. Unexpectedly, MEK1/2 inhibitors failed to enhance ara-C-mediated loss of mitochondrial membrane potential (DeltaPsi(m)), but instead induced substantial increases in cytosolic release of cytochrome c and Smac/DIABLO. U0126/ara-C-mediated apoptosis and pro-caspase 3 activation, but not cytochrome c or Smac/DIABLO release, were blocked by the pan-caspase inhibitor ZVAD-fmk. Together, these findings indicate that potentiation of ara-C-mediated lethality in HL-60 cells by MEK1/2 inhibitors involves enhanced cytosolic release of cytochrome c and Smac/DIABLO but not discharge of DeltaPsi(m), implicating activation of an apoptotic pathway that differs, at least with respect to the nature of the accompanying mitochondrial injury, from that triggered by ara-C alone.

A combinatorial approach for selectively inducing programmed cell death in human pancreatic cancer cells.

Pancreatic cancer is an extremely aggressive neoplasm whose incidence equals its death rate. Despite intensive analysis, the genetic changes that mediate pancreatic cancer development and effective therapies for diminishing the morbidity associated with this disease remain unresolved. Through subtraction hybridization, we have identified a gene associated with induction of irreversible growth arrest, cancer reversion, and terminal differentiation in human melanoma cells, melanoma differentiation associated gene-7 (mda-7). Ectopic expression of mda-7 when using a recombinant adenovirus, Ad.mda-7, results in growth suppression and apoptosis in a broad spectrum of human cancers with diverse genetic defects, without exerting deleterious effects in normal human epithelial or fibroblast cells. Despite the apparently ubiquitous antitumor effects of mda-7, pancreatic carcinoma cells are remarkably refractory to Ad.mda-7 induced growth suppression and apoptosis. In contrast, the combination of Ad.mda-7 with antisense phosphorothioate oligonucleotides, which target the K-ras oncogene (a gene that is mutated in 85 to 95% of pancreatic carcinomas), induces a dramatic suppression in growth and a decrease in cell viability by induction of apoptosis. In mutant K-ras pancreatic carcinoma cells, programmed cell death correlates with expression and an increase, respectively, in MDA-7 and BAX proteins and increases in the ratio of BAX to BCL-2 proteins. Moreover, transfection of mutant K-ras pancreatic carcinoma cells with an antisense K-ras expression vector and infection with Ad.mda-7 inhibits colony formation in vitro and tumorigenesis in vivo in nude mice. These intriguing observations demonstrate that a combinatorial approach, consisting of a cancer-specific apoptosis-inducing gene and an oncogene inactivation strategy, may provide the foundation for developing an effective therapy for pancreatic cancer.