Mutations in ERGIC1 cause Arthrogryposis multiplex congenita, neuropathic type.

Arthrogryposis multiplex congenita (AMC) is heterogeneous group of disorders characterized by non-progressive joint contractures from birth that involve more than 1 part of the body. There are various etiologies for AMC including genetic and environmental depends on the specific type, however, for most types, the cause is not fully understood. We previously reported large Israeli Arab kindred consisting of 16 patients affected with AMC neuropathic type, and mapped the locus to a 5.5 cM interval on chromosome 5qter. Using whole exome sequencing, we have now identified homozygous pathogenic variant in the ERGIC1 gene within the previously defined linked region. ERGIC1 encodes a cycling membrane protein which has a possible role in transport between endoplasmic reticulum and Golgi. We further show that this mutation was absent in more than 200 samples of healthy unrelated individuals of the Israeli Arab population. Thus, our findings expand the spectrum of hereditary AMC and suggest that abnormalities in protein trafficking may underlie AMC-related disorders.

Clinical features of conjoined lumbosacral nerve roots versus lumbar intervertebral disc herniations.

Unidentified nerve root anomalies, conjoined nerve root (CNR) being the most common, may account for some failed spinal surgical procedures as well as intraoperative neural injury. Previous studies have failed to clinically discern CNR from herniated discs and found their surgical outcomes as being inferior. A comparative study of CNR and disc herniations was undertaken. Between 2002 and 2008, 16 consecutive patients were diagnosed intraoperatively with CNR. These patients were matched 1:2 with 32 patients diagnosed with intervertebral disc herniations. Matching was done according to age (within 5 years), gender and level of pathology. Surgery for patients with CNR or disc herniations consisted of routine microsurgical techniques with microdiscectomy, hemilaminotomy, hemilaminectomy and foraminotomy as indicated. Outcomes were measured using the Oswestry Disability Index and the Short Form-36 Questionnaire. Clinical presentation, imaging studies and surgical outcomes were compared between the groups. Conjoined nerve root's incidence in this study was 5.8% of microdiscectomies performed. The S1 nerve root was mainly involved (69%), followed by L5 (31%). Patients with CNR tended to present with nerve root claudication (44%) compared to the radiculopathy accompanying disc herniations (75%). Neurologic deficit was less prevalent among patients with CNR. Nerve root tension tests were not helpful in distinguishing between the etiologies. Radiologist's suspicion threshold for nerve root anomalies was low (0%) and no coronal reconstructions were obtained. The surgeon's clinical suspicion accurately predicted 40% of the CNRs. Surgical outcomes did not differ between the cohorts regarding the rate of postoperative improvement, but CNR patients showed a trend toward having mildly worse long-term outcomes. Suspecting CNRs preoperatively is beneficial for appropriate treatment and avoiding the risk of intraoperative neural injury. With nerve root claudication and imaging suggestive of a "disc herniation", the surgeon should be alert to the differential diagnosis of a CNR. Treatment is directed at obtaining adequate decompression by laminectomy and foraminotomy to relieve the lateral recess stenosis. Outcomes can be expected to be similar to routine disc herniations.

The innate osteogenic potential of the maxillary sinus (Schneiderian) membrane: an ectopic tissue transplant model simulating sinus lifting.

Maxillary sinus membrane lifting is a common procedure aimed at increasing the volume of the maxillary sinus osseous floor prior to inserting dental implants. Clinical observations of bone formation in sinus lifting procedures without grafting bone substitutes were observed, but the biological nature of bone regeneration in sinus lifting procedures is unclear. This study tested whether this osteogenic activity relies on inherent osteogenic capacity residing in the sinus membrane by simulating the in vivo clinical condition of sinus lifting in an animal model. Maxillary sinus membrane cells were cultured in alpha-MEM medium containing osteogenic supplements (ascorbic acid, dexamethasone). Cultured cells revealed alkaline phosphatase activity and mRNA expression of osteogenic markers (alkaline phosphatase, bone sialoprotein, osteocalcin and osteonectin) verifying the osteogenic potential of the cells. Fresh tissue samples demonstrated positive alkaline phosphatase enzyme activity situated along the membrane-bone interface periosteum-like layer. To simulate the in vivo clinical conditions, the membranes were folded to form a pocket-like structure and were transplanted subcutaneously in immunodeficient mice for 8 weeks. New bone formation was observed in the transplants indicating the innate osteogenic potential within the maxillary Schneiderian sinus membrane and its possible contribution to bone regeneration in sinus lifting procedures.

Interaction index and different methods for determining drug interaction in combination therapy.

Studying and understanding the joint effect of combined treatments is important in pharmacology and in the development of combination therapies. The Loewe additivity model is one of the best general reference models for evaluating drug interactions. Based on this model, synergy occurs when the interaction index is less than one, while antagonism occurs when interaction index is greater than one. We expanded the meaning of the interaction index, and propose a procedure to calculate the interaction index and its associated confidence interval under the assumption that the dose-effect curve for a single agent follows Chou and Talalay's median effect equation. In addition, we review four response surface models based on the Loewe additivity model using a single parameter to determine drug interactions. We describe each of these models in the context of Loewe additivity model and discuss their relative advantages and disadvantages. We also provide S-PLUS/R code for each approach to facilitate the implementation of these commonly used methods.

Mechanisms of fenretinide-induced apoptosis.

Fenretinide, a synthetic retinoid, has emerged as a promising anticancer agent based on numerous in vitro and animal studies, as well as chemoprevention clinical trials. In vitro observations suggest that the anticancer activity of fenretinide may arise from its ability to induce apoptosis in tumor cells. Diverse signaling molecules including reactive oxygen species, ceramide, and ganglioside GD3 can mediate apoptosis induction by fenretinide in transformed, premalignant, and malignant cells. In many cell types, these signaling intermediates appear to be induced by mechanisms that are independent of retinoic acid receptor activation, and ultimately initiate the intrinsic or mitochondrial-mediated pathway of cell elimination. Numerous investigations conducted during the past 10 years have discovered a great deal about the apoptogenic activity of fenretinide. In this review we explore the mechanisms associated with fenretinide-induced apoptosis and highlight certain mechanistic underpinnings of fenretinide-induced cell death that remain poorly understood and thus warrant further characterization.

N-(4-hydroxyphenyl)retinamide-induced apoptosis triggered by reactive oxygen species is mediated by activation of MAPKs in head and neck squamous carcinoma cells.

N-(4-hydroxyphenyl)retinamide (4HPR), a synthetic retinoid effective in cancer chemoprevention and therapy, is thought to act via apoptosis induction resulting from increased reactive oxygen species (ROS) generation. As ROS can activate MAP kinases and protein kinase C (PKC), we examined the role of such enzymes in 4HPR-induced apoptosis in HNSCC UMSCC22B cells. 4HPR increased ROS level within 1 h and induced activation of caspase 3 and PARP cleavage within 24 h. Activation of MKK3/6 and MKK4, JNK, p38 and ERK was detected between 6 and 12 h, increased up to 24 h and preceded apoptosis. 4HPR-induced activation of these kinases was abrogated by the antioxidants BHA and vitamin C. SP600125, a JNK inhibitor, suppressed 4HPR-induced c-Jun phosphorylation, cytochrome c release from mitochondria and apoptosis. Suppression of JNK1 and JNK2 using siRNA decreased, whereas overexpression of wild type-JNK1 enhanced 4HPR-induced apoptosis. PD169316, a p38, inhibitor suppressed phosphorylation of Hsp27 and apoptosis. PD98059, an MEK1/2 inhibitor, also suppressed ERK1/2 activation and apoptosis induced by 4HPR. Likewise, PKC inhibitor GF109203X suppressed ERK and p38 phosphorylation and PARP cleavage. These data indicate that 4HPR-induced apoptosis is triggered by ROS increase, leading to the activation of the mitogen-activated protein serine/threonine kinases JNK, p38, PKC and ERK, and subsequent apoptosis.

Haptoglobin and the development of cerebral artery vasospasm after subarachnoid hemorrhage.

BACKGROUND: Vasospasm is a prolonged constriction of a cerebral artery that is induced by hemoglobin after subarachnoid hemorrhage (SAH). The subarachnoid blood clot also contains the protein haptoglobin, which acts to neutralize hemoglobin. Because the haptoglobin alpha gene is dimorphic, a person can expresses only one of three types of haptoglobin (alpha1-alpha1, alpha1-alpha2, or alpha2-alpha2) depending on the alpha subunit genes he or she inherits. Each of these three haptoglobin types has different antihemoglobin activities; therefore, haptoglobin may influence the development of vasospasm differently in various patients with SAH. OBJECTIVE: To determine whether SAH patients who have haptoglobin containing the alpha2 subunit would be more likely to develop vasospasm than would be SAH patients who have haptoglobin alpha1-alpha1. METHODS AND RESULTS: A total of 32 patients with Fisher Grade 3 SAH were enrolled in this study. Haptoglobin type was determined by polyacrylamide gel electrophoresis. The primary measure for vasospasm was increased blood flow velocities as detected by daily transcranial Doppler ultrasonography (TCD). The authors found that only 2 of 9 patients with haptoglobin alpha1-alpha1 (22%) had development of "possible" vasospasm as measured by TCD, whereas 20 of 23 patients with the haptoglobin alpha2 subunit (either the alpha1-alpha2 or alpha2-alpha2 haptoglobin types) had development of "possible" vasospasm (87%). The secondary measure for vasospasm was cerebral angiography performed between 3 and 14 days after SAH. Similar results (17% vs 56%) were seen between these groups in those patients who underwent cerebral angiography, although its inconsistent use limited the strength of the statistical comparison. CONCLUSIONS: Haptoglobins containing the alpha2 subunit seem to be associated with a higher rate of vasospasm than is haptoglobin alpha1-alpha1.

Global detection of molecular changes reveals concurrent alteration of several biological pathways in nonsmall cell lung cancer cells.

To identify the molecular changes that occur in non-small cell lung carcinoma (NSCLC), we compared the gene expression profile of the NCI-H292 (H292) NSCLC cell line with that of normal human tracheobronchial epithelial (NHTBE) cells. The NHTBE cells were grown in a three-dimensional organotypic culture system that permits maintenance of the normal pseudostratified mucociliary phenotype characteristic of bronchial epithelium in vivo. Microarray analysis using the Affymetrix oligonucleotide chip U95Av2 revealed that 1,683 genes showed a >1.5-fold change in expression in the H292 cell line relative to the NHTBE cells. Specifically, 418 genes were downregulated and 1,265 were upregulated in the H292 cells. The expression data for selected genes were validated in several different NSCLC cell lines using quantitative real-time PCR and Western analysis. Further analysis of the differentially expressed genes indicated that WNT responses, apoptosis, cell cycle regulation and cell proliferation were significantly altered in the H292 cells. Functional analysis using fluorescence-activated cell sorting confirmed concurrent changes in the activity of these pathways in the H292 line. These findings show that (1) NSCLC cells display deregulation of the WNT, apoptosis, proliferation and cell cycle pathways, as has been found in many other types of cancer cells, and (2) that organotypically cultured NHTBE cells can be used as a reference to identify genes and pathways that are differentially expressed in tumor cells derived from bronchogenic epithelium.

ICAM-5 (telencephalin) gene expression in head and neck squamous carcinoma tumorigenesis and perineural invasion!

ICAM-5 (telencephalin) is an intercellular adhesion molecule reported to be expressed only in the somatodendritic membrane of telencephalic neurons. We recently identified high ICAM-5 expression in a cDNA array study of head and neck neoplasms with a propensity for perineural invasion. To determine the association of this gene in tumorigenesis and perineural invasion, we analyzed the expression and functional status of ICAM-5 mRNA transcripts in 30 different human cancer cell lines and 25 head and neck squamous carcinoma specimens by reverse-transcriptase polymerase chain reaction (cell lines and specimens) and in vitro functional assays (cell lines). ICAM-5 transcripts were detected in 28 (93%) of 30 cell lines derived from primary head and neck, colon, thyroid, cervical, pancreatic, skin, and adenoid cystic carcinomas. In cell lines, small interfering RNA blocked ICAM-5 expression and inhibited cell proliferation. Treatment with the phosphatidylinositol 3'-kinase (PBK) inhibitor LY294002 resulted in ICAM-5 down-regulation. In tissue specimens, none of the 25 histologically normal oral mucosal specimens had detectable ICAM-5 level, whereas 16 (64%) of the 25 matched primary squamous carcinomas showed expression. Carcinoma specimens high ICAM-5 expression had a high incidence of perineural invasion. Our study indicates that ICAM-5 may play a role in tumorigenesis and perineural invasion, most likely through the P13K/Akt-signaling pathway.

Apoptosis induction by the natural product cancer chemopreventive agent deguelin is mediated through the inhibition of mitochondrial bioenergetics.

Deguelin exhibits chemopreventive properties in animal carcinogenesis models. The mechanism underpinning the chemopreventive effects of deguelin has not been fully elucidated. However, it has been suggested that this agent reduces ornithine decarboxylase activity, and perhaps the activity of other signaling intermediates associated with tumorigenesis, by inhibiting mitochondrial bioenergetics. We sought to determine if deguelin could trigger apoptosis by inhibiting mitochondrial bioenergetics. Therefore, we compared and contrasted the effects of deguelin on cells from two human cutaneous squamous cell carcinoma cell lines (parental cells) and their respiration-deficient clones lacking mitochondrial DNA (rho0). While deguelin promoted marked apoptosis in the parental cells in a dose- and time-dependent manner, it failed to do so in the rho0 clones. Furthermore, short-term exposure to deguelin diminished oxygen consumption by the parental cells and promoted mitochondrial permeability transition as evidenced by the dissipation of mitochondrial inner transmembrane potential, reactive oxygen species production, cardiolipin peroxidation, caspase activation, and mitochondrial swelling. Mitochondrial permeability transition was not observed in the rho0 clones exposed to deguelin. These results demonstrate that deguelin induces apoptosis in skin cancer cells by inhibiting mitochondrial bioenergetics and provide a novel mechanism for the putative anticancer activity of this agent.

Involvement of c-Fos in fenretinide-induced apoptosis in human ovarian carcinoma cells.

Fenretinide (HPR), a synthetic retinoid that exhibits lower toxicity than other retinoids, has shown preventive and therapeutic activity against ovarian tumors. Although the growth inhibitory effects of HPR have been ascribed to its ability to induce apoptosis, little is known about the molecular mechanisms involved. Since the proto-oncogene c-Fos has been implicated in apoptosis induction, we analyzed its role in mediating HPR response in a human ovarian carcinoma cell line (A2780) sensitive to HPR apoptotic effect. In these cells, HPR treatment caused induction of c-Fos expression, whereas such an effect was not observed in cells made resistant to HPR-induced apoptosis (A2780/HPR). Moreover, in a panel of other human ovarian carcinoma cell lines, c-Fos inducibility and HPR sensitivity were closely associated. Ceramide, which is involved in HPR-induced apoptosis, was also involved in c-Fos induction because its upregulation by HPR was reduced by fumonisin B(1), a ceramide synthase inhibitor. The causal relationship between c-Fos induction and apoptosis was established by the finding of an increased apoptotic rate in cells overexpressing c-Fos. Similarly to that observed for c-Fos expression, HPR treatment increased c-Jun expression in HPR-sensitive but not in HPR-resistant cells, suggesting the involvement of the transcription factor activating protein 1 (AP-1) in HPR-induced apoptosis. In gene reporter experiments, HPR stimulated AP-1 transcriptional activity and potentiated the AP-1 activity induced by 12-tetradecanoylphorbol 13-acetate. Furthermore, inhibition of AP-1 DNA binding, by transfecting A2780 cells with a dominant-negative Fos gene, caused decreased sensitivity to HPR apoptotic effects. Overall, the results indicate that c-Fos plays a role in mediating HPR-induced growth inhibition and apoptosis in ovarian cancer cells and suggest that c-Fos regulates these processes as a member of the AP-1 transcription factor.

Receptor-independent induction of apoptosis by synthetic retinoids.

Retinoids modulate cell proliferation, differentiation and apoptosis. Many of these effects are mediated by nuclear retinoid receptors. However, studies with certain synthetic retinoids, including some that can activate retinoid receptors, revealed that they affect cell growth and especially apoptosis by mechanisms that are independent of nuclear receptors. This chapter describes the pro-apoptotic effects of the synthetic retinoid CD437 [6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid], and structurally-related retinoids and summarizes the mechanisms by which they induce apoptosis.

A randomized clinical trial of 4-hydroxyphenylretinamide for high-grade squamous intraepithelial lesions of the cervix.

PURPOSE: Previous trials of topical trans-retinoic acid treatment of cervical intraepithelial neoplasia (CIN) grades 2 and 3 led to a statistically significant regression of CIN 2, but not CIN 3. We tested N-(4-hydroxyphenyl)retinamide (4-HPR), a promising oral retinoid that has been shown to induce apoptosis through nonretinoic receptor acid-mediated pathways, for its toxicity and efficacy against CIN 2/3. EXPERIMENTAL DESIGN: In a blinded randomized trial, 4-HPR at 200 mg/day for 6 months (with a 3-day/month drug holiday) was compared with placebo in patients with biopsy-proven CIN-2/3 [high-grade squamous intraepithelial lesions (HGSILs)]. Patients were treated with placebo or 4-HPR for 6 months, biopsied, and then followed for an additional 6 months. At the 12-month end point, they underwent either loop excision if a histological lesion was present or a biopsy from the original area of the lesion if no lesion was present. RESULTS: An interim analysis of blinded data showed a significantly worse prognosis at 12 months for one group. When the code was broken because of the poorer outcomes, we discovered that the 4-HPR treatment arm was performing more poorly than was the placebo at 6 and 12 months (25 versus 44% response rates at 6 months; 14 versus 50% at 12 months). Toxicity was not significant in either arm. CONCLUSIONS: 4-HPR at 200 mg/day with a 3-day/month drug holiday is not active compared with placebo in the treatment of HGSIL. Because 4-HPR is active in the laboratory, the lack of effect in our trial may indicate that higher doses are needed in patients to achieve comparable results.

Differential effects of chromosome 3p deletion on the expression of the putative tumor suppressor RAR beta and on retinoid resistance in human squamous carcinoma cells.

Retinoids' effects on cell growth and differentiation are mediated by nuclear retinoid receptors, which are ligand-activated transcription enhancing factors. Because the expression of the retinoic acid receptor beta (RARbeta) gene, which is located on chromosome 3p24, is diminished in premalignant and malignant tissues it has been proposed that it acts as a tumor suppressor. To test the hypothesis that RARbeta loss leads to retinoid resistance, we studied several karyotyped head and neck squamous carcinoma (HNSCC) cell lines (UMSCC-17A, -17B, -22A, -22B, and -38) with deletion of one chromosome 3p arm. RARbeta mRNA was neither detected nor induced by retinoic acid in these cells, whereas it was expressed and induced by retinoic acid in two other HNSCC cell lines (1483 and 183) without 3p deletion. Methylation of the RARbeta gene promoter was detected in the 17B and 22B cells that failed to express RARbeta but no methylation was found in 183A cells that did express RARbeta mRNA. Responsiveness of HNSCC cells to several retinoids in assays of growth inhibition and colony formation, was rank ordered as: 22B>1483>38>183>17B. Additionally, retinoid response elements were transactivated in 22B more efficiently than in 17B cells. These results indicate that loss of RARbeta expression does not necessarily lead to loss of growth inhibition by retinoids or to a block of retinoid signaling.

Diminished expression of S100A2, a putative tumor suppressor, at early stage of human lung carcinogenesis.

To identify and understand early events in lung carcinogenesis, we used a cDNA array to screen for genes that are expressed differentially in normal human bronchial epithelial (NHBE) cells and a tumorigenic cell line (1170-I) derived from immortalized HBE cells after exposure to cigarette smoke condensate in vivo. Among these genes, we have identified the S100A2 gene, which encodes a nuclear calcium-binding protein, as being down-regulated in the 1170-I cells. Because this gene has been implicated as a tumor suppressor in breast cancer, we examined its potential role as a tumor suppressor in lung carcinogenesis. Levels of S100A2 transcript and protein, which were high in NHBE cells, decreased by up to 50% in immortalized HBE cells (BEAS-2B and 1799) and to low to nearly undetectable levels in transformed (1198) and tumorigenic (1170-I) HBE cells. Furthermore, S100A2 mRNA and protein were undetectable in 8 and expressed at a reduced level in 3 of 11 non-small cell lung cancer (NSCLC) cell lines. Positive immunohistochemical staining of S100A2 was detected in the majority (75-83%) of normal and hyperplastic lung tissues, whereas it was detected in <10% of metaplastic lung tissues, squamous cell carcinoma, and adenocarcinoma. Treatment of 1170-I HBE and NSCLC cells with 5-aza-2'-deoxycytidine resulted in partial restoration of S100A2 expression in seven of eight cell lines. Indeed, CpG methylation was detected in the promoter region of the S100A2 gene. Our results suggest that S100A2 expression is suppressed early during lung carcinogenesis, possibly by hypermethylation of its promoter, and that its loss may be a contributing factor in lung cancer development or a biomarker of early changes in this process.

Inactivation of retinoic acid receptor beta by promoter CpG hypermethylation in gastric cancer.

Inactivation of nuclear retinoic acid receptor beta (RARbeta) expression is implicated in tumorigenesis. We hypothesized that loss of RARbeta in gastric cancer cells may occur as a result of multiple factors, including epigenetic modifications which alter RARbeta promoter chromatin structure. We examined hypermethylation of CpG islands present in the RARbeta promoter by methylation-specific PCR and the expression of RARbeta in gastric cancer cell lines and tissues. Three (MKN-28, -45 and -74) out of eight gastric cancer cell lines had a loss of RAR expression associated with promoter methylation. RARbeta expression was retrieved in these cell lines by treatment with 5-azacytidine or by the histone deacetylase inhibitor trichostatin A. Promoter hypermethylation was detected in 64% (7/11) of gastric carcinoma tissues with reduced expression of RARbeta, whereas it was detected in 22% (2/9) of tumors with retained RARbeta expression. To investigate the functions of exogenous RARbeta in gastric cancer cells, we transfected a retroviral RARbeta expression vector (LNSbeta) into MKN-28 cells that have hypermethylation of the RARbeta promoter. Overexpression of RAR in MKN-28 cells appeared to regulate the expression of DNA methyltransferase and DNA demethylase and the acetylation of hitone H4. These results suggest that the transcriptional inactivation of the RARbeta by promoter CpG hypermethylation is frequently associated with gastric carcinoma. Our data also suggests that DNA methylation plays a pivotal role in establishing and maintaining an inactive state of RARbeta by rendering the chromatin structure inaccessible to the transcription machinery.

All-trans-retinoic acid enhances the effect of adenovirus-mediated wild-type p53 gene transfer in head and neck squamous cell carcinoma.

OBJECTIVES: Adenovirus-mediated p53 (AdCMVp53) gene therapy for cancer is currently undergoing phase III clinical trials. One problematic aspect of this therapy is that the current protocols result in low transduction of the therapeutic virus in vivo. To search new modalities that can enhance the effect of AdCMVp53 gene therapy, we focused on retinoids. METHODS: To study the effect of ATRA in combination with AdCMVp53 gene therapy, we pretreated head and neck squamous cell carcinoma (HNSCC) cells for 72 hours with a low-dose All-trans-retinoic acid (ATRA) (10-7 M-10-8 M) which will not affect the in vitro cell growth, and then infected the cells with low MOI (30MOI) AdCMVp53. In vitro cell proliferation assays, cell cycle assays were performed. Expression of p53 and p53-related gene products, BAX and p21, were examined. RESULTS: The combined treatment with ATRA and Ad-p53 suppressed cell growth and induced apoptosis significantly more than AdCMVp53 treatment alone (P <.05). p53 expression significantly increased more after the combined treatment than after either treatment alone, at both the transcription and protein levels. In addition, increased expression of p21 and BAX, which are downstream gene products of p53, was observed in the combination. ATRA also enhanced the expression of green fluorescent protein (GFP) transduced by an adenovirus-cytomegalovirus (CMV)-GFP vector suggesting ATRA enhances adenovirus-CMV-promoted vectors through transcription. CONCLUSIONS: Our results indicate that ATRA enhances AdCMVp53 expression through transcriptional mechanisms and can synergistically induce apoptosis in HNSCC cells. ATRA has a potential to enhance the effect of adenovirus-mediated p53 gene therapy for HNSCC.

Evidence supporting a role for mitochondrial respiration in apoptosis induction by the synthetic retinoid CD437.

Retinoids have been shown to modulate cell proliferation, differentiation, and apoptosis. It is thought that these effects mediate the chemopreventive and therapeutic effects of retinoids. Recently, some synthetic retinoids, including 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437), have been found to induce apoptosis even in tumor cell lines that are resistant to all-trans retinoic acid. The proapoptotic activity of CD437 has been attributed to mitochondrial dysfunction via the induction of mitochondrial permeability transition (P. Marchetti et al., Cancer Res. 59: 6257-6266, 1999). The mechanistic aspects pertaining to how CD437 promotes changes in mitochondrial function are unclear. This study investigated the role of mitochondrial respiration in CD437-induced apoptosis. Human cutaneous squamous cell carcinoma COLO 16 cells were chronically exposed to ethidium bromide to inhibit mitochondrial DNA synthesis and produce respiration-deficient clones. These clones were exposed to CD437 (

Mitochondrial respiration is uniquely associated with the prooxidant and apoptotic effects of N-(4-hydroxyphenyl)retinamide.

The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) is being examined in both chemoprevention and therapy clinical trials. Yet, its mechanism(s) of action is still not fully elucidated. In previous studies, an increase in mitochondrial reactive oxygen species has been proposed as one mechanism through which 4HPR could exert its proapoptotic effects. This study explored whether mitochondrial respiration is required for 4HPR action using human cutaneous squamous cell carcinoma cells and respiration-deficient clones. In parental cells, 4HPR rapidly promoted hydroperoxide production followed by mitochondrial permeability transition, caspase activity, and DNA fragmentation. Short term exposure to 4HPR also inhibited oxygen consumption in parental cells. This activity was reversed by the antioxidant vitamin C indicating the prooxidant effect of 4HPR directly impaired mitochondrial function. In respiration-deficient clones, the proapoptotic qualities of 4HPR were conspicuously diminished illustrating a central role for mitochondrial respiration in 4HPR-induced cell death. In parental cells, various mitochondrial inhibitors were examined to determine potential sites associated with the prooxidant activity of 4HPR. Inhibitors of Complex II as well as center i inhibitors of Complex III enhanced 4HPR-induced hydroperoxide production. Complex I inhibitors, center o inhibitors of Complex III, cyanide, oligomycin A, and coenzyme Q analogues decreased 4HPR-induced hydroperoxide production. The coenzyme Q analogues were very effective in this respect, and they also blocked the enhanced hydroperoxide production obtained when center i inhibitors were combined with 4HPR. These results suggest the prooxidant property of 4HPR is associated with redox metabolism via an enzymatic process occurring at a quinone-binding site in Complex I and/or center o of Complex III.