David M. Hockenbery

Bcl-2 family proteins as regulators of oxidative stress

The Bcl-2 family of proteins includes pro- and anti-apoptotic factors acting at mitochondrial and microsomal membranes. An impressive body of published studies, using genetic and physical reconstitution experiments in model organisms and cell lines, supports a view of Bcl-2 proteins as the critical arbiters of apoptotic cell death decisions in most circumstances (excepting CD95 death receptor signaling in Type I cells). Evasion of apoptosis is one of the hallmarks of cancer [Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57-70], relevant to tumorigenesis as well as resistance to cytotoxic drugs, and deregulation of Bcl-2 proteins is observed in many cancers [Manion MK, Hockenbery DM. Targeting BCL-2-related proteins in cancer therapy. Cancer Biol Ther. 2003;2:S105-14; Olejniczak ET, Van Sant C, Anderson MG, Wang G, Tahir SK, Sauter G, et al. Integrative genomic analysis of small-cell lung carcinoma reveals correlates of sensitivity to bcl-2 antagonists and uncovers novel chromosomal gains. Mol Cancer Res. 2007;5:331-9]. The rekindled interest in aerobic glycolysis as a cancer trait raises interesting questions as to how metabolic changes in cancer cells are integrated with other essential alterations in cancer, e.g. promotion of angiogenesis and unbridled growth signals. Apoptosis induced by multiple different signals involves loss of mitochondrial homeostasis, in particular, outer mitochondrial membrane integrity, releasing cytochrome c and other proteins from the intermembrane space. This integrative process, controlled by Bcl-2 family proteins, is also influenced by the metabolic state of the cell. In this review, we consider the role of reactive oxygen species, a metabolic by-product, in the mitochondrial pathway of apoptosis, and the relationships between Bcl-2 functions and oxidative stress.

Oral beclomethasone dipropionate for treatment of intestinal graft-versus-host disease: A randomized, controlled trial

BACKGROUND & AIMSnBeclomethasone dipropionate (BDP), a topically active steroid, seemed to be an effective treatment for intestinal graft-versus-host disease (GVHD) in a phase I study. The aim of this study was to compare the effectiveness of oral BDP to that of placebo capsules in treatment of intestinal GVHD.nnnMETHODSnSixty patients with anorexia and poor oral intake because of intestinal GVHD were randomized to receive prednisone (1 mg.kg-1.day-1) plus either oral BDP (8 mg/day) or placebo capsules. Initial responders who were eating at least 70% of caloric needs at evaluation on day 10 continued to take study capsules for an additional 20 days while the prednisone dose was rapidly tapered. The primary end point was the frequency of a durable treatment response at day 30 of treatment.nnnRESULTSnThe initial treatment response at day 10 was 22 of 31 (71%) in the BDP/prednisone group vs. 16 of 29 (55%) for the placebo/prednisone group. The durable treatment response at day 30 was 22 of 31 (71%) vs. 12 of 29 (41%), respectively (P = 0.02).nnnCONCLUSIONSnThe combination of oral BDP capsules and prednisone was more effective than prednisone alone in treating intestinal GVHD. Oral BDP allowed prednisone doses to be rapidly tapered without recurrent intestinal symptoms.

Induction of Apoptosis Using Inhibitors of Lysophosphatidic Acid Acyltransferase-β and Anti-CD20 Monoclonal Antibodies for Treatment of Human Non-Hodgkin's Lymphomas

Purpose: Lysophosphatidic acid acyltransferase-β (LPAAT-β) is a transmembrane enzyme critical for the biosynthesis of phosphoglycerides whose product, phosphatidic acid, plays a key role in raf and AKT/mTor-mediated signal transduction. Experimental Design: LPAAT-β may be a novel target for anticancer therapy, and, thus, we examined the effects of a series of inhibitors of LPAAT-β on multiple human non–Hodgkins lymphoma cell lines in vitro and in vivo. Results: We showed that five LPAAT-β inhibitors at doses of 500 nmol/L routinely inhibited growth in a panel of human lymphoma cell lines in vitro by >90%, as measured by [3H]thymidine incorporation. Apoptotic effects of the LPAAT-β inhibitors were evaluated either alone or in combination with the anti-CD20 antibody, Rituximab. The LPAAT-β inhibitors induced caspase-mediated apoptosis at 50 to 100 nmol/L in up to 90% of non–Hodgkins lymphoma cells. The combination of Rituximab and an LPAAT-β inhibitor resulted in a 2-fold increase in apoptosis compared with either agent alone. To assess the combination of Rituximab and a LPAAT-β inhibitor in vivo, groups of athymic mice bearing s.c. human Ramos lymphoma xenografts were treated with the LPAAT-β inhibitor CT-32228 i.p. (75 mg/kg) daily for 5 d/wk × 4 weeks (total 20 doses), Rituximab i.p. (10 mg/kg) weekly × 4 weeks (4 doses total), or CT-32228 plus Rituximab combined. Treatment with either CT-32228 or Rituximab alone showed an approximate 50% xenograft growth delay; however, complete responses were only observed when the two agents were delivered together. Conclusions: These data suggest that Rituximab, combined with a LPAAT-β inhibitor, may provide enhanced therapeutic effects through apoptotic mechanisms.

Severe gastrointestinal bleeding after hematopoietic cell transplantation, 1987-1997: incidence, causes, and outcome.

Severe gastrointestinal bleeding after hematopoietic cell transplantation, 1987–1997: incidence, causes, and outcome

Investigating neoplastic progression of ulcerative colitis with label-free comparative proteomics

Patients with extensive ulcerative colitis (UC) have an increased risk of colorectal cancer. Although UC patients generally undergo lifelong colonoscopic surveillance to detect dysplasia or cancer in the colon, detection of cancer in this manner is expensive and invasive. An objective biomarker of dysplasia would vastly improve the clinical management of cancer risk in UC patients. In the current study, accurate mass and time methods with ion intensity-based label-free proteomics are applied to profile individual rectal and colon samples from UC patients with dysplasia or cancer (UC progressors) compared to rectal samples from patients that are dysplasia/cancer free (UC nonprogressors) to identify a set of proteins in the rectum mucosa that differentiate the two groups. In addition to the identification of proteins in UC dysplastic colon tissue, we for the first time identified differentially expressed proteins in nondysplastic rectal tissue from UC progressors. This provides a candidate pool of biomarkers for dysplasia/cancer that could be detected in a random nondysplastic rectal biopsy. Mitochondrial proteins, cytoskeletal proteins, RAS superfamily, proteins relating to apoptosis and metabolism were important protein clusters differentially expressed in the nondysplastic and dysplastic tissues of UC progressors, suggesting their importance in the early stages of UC neoplastic progression. Among the differentially expressed proteins, immunohistochemistry analysis confirmed that TRAP1 displayed increased IHC staining in UC progressors, in both dysplastic and nondysplastic tissue, and CPS1 showed a statistically significant difference in IHC staining between the nonprogressor and progressor groups. Furthermore, rectal CPS1 staining could be used to predict dysplasia or cancer in the colon with 87% sensitivity and 45% specificity, demonstrating the feasibility of using surrogate biomarkers in rectal biopsies to predict dysplasia and/or cancer in the colon.

Prevention of Fas-mediated hepatic failure by transferrin

Recent studies in lymphohemopoietic cells show that transferrin (Tf), a pivotal component of iron transport and metabolism, also exerts cytoprotective functions. We show here in a murine model that Tf interferes with Fas-mediated hepatocyte death and liver failure. The mechanism involves the downregulation of apoptosis via BID, cytochrome c, caspase-3 and caspase-9, and upregulation of antiapoptotic signals via Bcl-xL. The results obtained with iron-saturated Tf, Apo-Tf and the iron-chelator salicylaldehyde isonicotinoyl hydrazone indicate that the observed antiapoptotic effect of Tf was not mediated by iron alone. In conclusion, the data suggest that Tf has broader functions than previously recognized and may serve as a cytoprotective agent.

Differential responses of FLIPLong and FLIPShort-overexpressing human myeloid leukemia cells to TNF-α and TRAIL-initiated apoptotic signals

OBJECTIVEnClonal marrow cells from patients with early myelodysplastic syndrome (MDS) undergo apoptosis in response to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). Cells from advanced MDS are resistant to TRAIL. Two isoforms of the Flice inhibitory protein (FLIP) short (FLIPS) and FLIP long (FLIPL), which modulate TRAIL signals, showed disease-stage-dependent differential regulation. Therefore, we aimed at characterizing potential differential effects of FLIPL and FLIPS, on TRAIL and TNF-alpha-induced apoptosis in model leukemic cell lines.nnnMATERIALS AND METHODSnUsing lentiviral constructs, FLIPL and FLIPS, as well as a green fluorescent protein control were overexpressed in ML-1 cells, which constitutively express very low levels of FLIP and are highly sensitive to apoptosis induction. Cells were then exposed to TRAIL or TNF-alpha, and effects on the extrinsic and intrinsic pathways of apoptosis induction were assessed.nnnRESULTSnOverexpression of FLIP reduced TRAIL and TNF-alpha-induced apoptosis in ML-1 cells. However, while FLIPL completely abrogated apoptosis, FLIPS allowed for BID cleavage and caspase-3 activation. Concurrently, there was a decline of Bcl-xL and X-linked inhibitor of apoptosis protein (XIAP) in FLIPS cells followed by apoptosis. Further, inhibition of nuclear factor-kappaB (NF-kappaB) activation in TNF-alpha-treated cells resulted in profound apoptosis in FLIPS, but not in FLIPL-overexpressing cells, consistent with the observations in patients with early stage MDS. Inhibition of NF-kappaB had only minimal effects on TRAIL signaling.nnnCONCLUSIONnThus, FLIPL and FLIPS exerted differential effects in myeloid leukemic cell lines in response to TRAIL and TNF-alpha. It might be possible to therapeutically exploit those differences with effector molecules specific for the FLIP isoforms.

Lymphocytic gastritis resembling graft-vs.-host disease following autologous hematopoietic cell transplantation

Although cutaneous graft-vs.-host disease (GVHD) has been noted after autologous hematopoietic cell transplantation, intestinal involvement has not been well documented. We evaluated 197 patients undergoing autologous transplantation for intestinal symptoms; the source for hematopoietic cells was marrow (n=32), peripheral blood stem cells (n=146), or both (n=19). Patients with persistent nausea, vomiting, and anorexia after day 20 underwent upper intestinal endoscopy and mucosal biopsy. Eight patients (4.1%) had diffuse edema, erythema of gastric mucosa, and histological evidence of lymphocytic gastritis with focal apoptosis of crypt epithelial cells-typical of the findings in acute GVHD. All studies for viral, fungal, or bacterial causes were negative. Two patients showed evidence of GVHD in skin and liver, respectively. All patients received 1 mg/kg/day of oral prednisone for 10 days; symptomatic improvement often occurred within days of therapy onset. At the end of corticosteroid treatment, complete resolution of symptoms was seen in all eight patients. In one patient, elevated serum alkaline phosphatase levels gradually normalized over the ensuing 3-4 weeks. When followed up 3 months after treatment, all patients remained symptom-free without evidence of recurrent intestinal symptoms. We concluded that recipients of autologous hematopoietic cells may develop intestinal symptoms caused by a lymphocytic gastritis that is typical of acute GVHD. Patients with this syndrome promptly responded to treatment of a short course of prednisone. The pathogenesis of gastric epithelial damage after autologous transplant is unknown.

Response of Steroid-Refractory Acute GVHD to α1-Antitrypsin

α1-Antitrypsin (AAT) is a serine protease inhibitor with anti-inflammatory, antiapoptotic, and immunomodulatory properties. It has therapeutic efficacy in animal models of autoimmune diseases, inflammatory disorders, and transplantation. In a phase I/II open-label single-center study, we administered AAT (Glassia; Baxalta/Kamada, New Ziona, Israel) as salvage therapy to 12 patients with steroid-refractory acute graft-versus-host disease (GVHD). AAT was given i.v. at 2 dose levels over a 15-day course. All patients had grades III or IV GVHD with stage 4 gut involvement. After treatment, plasma AAT levels increased in both cohorts and remained within 2 to 4xa0mg/mL for the duration of treatment. No clinically relevant toxicities attributable to AAT were observed. GVHD manifestations improved in 8 of 12 patients, and 4 responses were complete. Six patients (50%) were alive at last follow-up (>104 to >820xa0days). These findings show that AAT is well tolerated and has efficacy in the treatment of steroid-refractory severe acute GVHD. Further studies are warranted.

Highlights of the National Cancer Institute Workshop on Mitochondrial Function and Cancer

This workshop was stimulated by the desire of the National Cancer Institute to examine various aspects of mitochondrial function as they relate to tumorigenesis, apoptosis, and cancer therapy. Through endosymbiosis, a bacterial ancestor took its position in the eukaryotic cytoplasm and serves as the