The Summer School Oncology Groningen: Improving a Successful International Course by Refining the Old, Maintaining What's Good.

For more than two decades, the International Summer School Oncology for Medical Students (ISOMS) has organized a biennial 2-week international summer school program in Groningen, the Netherlands. The summer school aims to increase knowledge about general cancer care, reduce fear of talking to cancer patients, and expose students to cancer-related problems. After 22 years, there was a need to improve the summer school format, the application procedure, and the intensity of the course. Here, we describe and evaluate these and additional changes that were made to the program. Several changes were made to the summer school format. The course was shortened from 10 days to a more intensive 7 days. The scientific program was integrated with the clinical program and students were taught scientific writing and presentation skills. The application process involved a personal video pitch. Importantly, the new summer school format was organized by a committee in which medical students had the lead. To evaluate the changes to the summer school, we conducted knowledge tests and regularly obtained feedback. There was a high overall student satisfaction, with a median score of a 9 out of 10. Students appreciated the interactive sessions and practicals and the scientific program, and were satisfied with the course level. All students had improved test scores. Improvement points highlighted the need for a less packed schedule and more lectures on basic oncology principles, or were related to specific lectures. The student-led innovation and adaptation of the ISOMS has been successful.

SSTR-2 as a potential tumour-specific marker for fluorescence-guided meningioma surgery.

BACKGROUND: Meningiomas are the most frequently occurring primary intracranial tumours in adults. Surgical removal can only be curative by complete resection; however surgical access can be challenging due to anatomical localization and local invasion of bone and soft tissues. Several intraoperative techniques have been tried to improve surgical resection, including intraoperative fluorescence guided imaging; however, no meningioma-specific (fluorescent) targeting has been developed yet. Here, we aimed to identify the most promising biomarkers for targeted intra-operative fluorescence guided meningioma surgery. METHODS: One hundred forty-eight meningioma specimens representing all meningioma grades were analysed using immunohistochemistry (IHC) on tissue microarrays (TMAs) to determine expression patterns of meningioma biomarkers epithelial membrane antigen (EMA), platelet-derived growth factor ß (PDGF-ß), vascular endothelial growth factor α (VEGF-α), and somatostatin receptor type 2 (SSTR-2). Subsequently, the most promising biomarker was selected based on TArget Selection Criteria (TASC). Marker expression was examined by IHC in 3D cell culture models generated from freshly resected tumour material. RESULTS: TMA-IHC showed strongest staining for SSTR-2. All cases were positive, with 51.4% strong/diffuse, 30.4% moderate/diffuse and only 18.2% focal/weak staining patterns. All tested biomarkers showed at least weak positivity in all meningiomas, regardless of WHO grade. TASC analysis showed that SSTR-2 was the most promising target for fluorescence guided imaging, with a total score of 21 (out of 22). SSTR-2 expression was determined on original patient tumours and 3D cultures of three established cultures. CONCLUSIONS: SSTR-2 expression was highly sensitive and specific in all 148 meningiomas, regardless of WHO grade. According to TASC analysis, SSTR-2 is the most promising receptor for meningioma targeting. After establishing in vitro meningioma models, SSTR-2 cell membrane expression was confirmed in two of three meningioma cultures as well. This indicates that specific fluorescence in an experimental setting can be performed for the further development of targeted fluorescence guided meningioma surgery and near-infrared fluorescent tracers targeting SSTR-2.

Microenvironment involved in FPR1 expression by human glioblastomas.

Formyl peptide receptor 1 (FPR1) activity in U87 glioblastoma (GBM) cells contributes to tumor cell motility. The present study aimed to evaluate the FPR1 expression in human GBM, the possibility to elicit agonist induced FPR1 activation of GBM cells and inhibit this activation with chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). Immunohistochemistry was used to assess FPR1 expression in GBM patient samples, which was present in all 178 samples. Also FPR1 mRNA levels measured with quantitative PCR, could be detected in all 25 GBM patient samples tested. Activation of FPR1 in U87 cells, as measured by human mitochondrial-derived agonists, increased calcium mobilization, AKT and ERK1/2 phosphorylation, and ligand-induced migration. Inhibition of all responses could be achieved with CHIPS. Eight early passage human Groningen Glioma (GG) cell lines, isolated from primary GBM tissue were screened for the presence of FPR1. FPR1 mRNA and protein expression as well as receptor activation could not be detected in any of these early passage GG cell lines. However FPR1 was present in ex vivo tumors formed by the same GG cell lines after being implanted in mouse brains. FPR1 is highly expressed in human GBM specimens, it can be activated by human mitochondrial-derived agonists in U87 and inhibited with CHIPS. FPR1 cannot be detected in early passage GG cell lines in vitro, however when engrafted in the mouse brain these cells show FPR1 expression. These results suggest a role of the brain microenvironment in FPR1 expression in GBM.

Nutlin-3 preferentially sensitises wild-type p53-expressing cancer cells to DR5-selective TRAIL over rhTRAIL.

BACKGROUND: Tumour cell-selective activation of apoptosis by recombinant human TNF-related apoptosis-inducing ligand (rhTRAIL) is enhanced through co-activation of p53 by chemotherapeutic drugs. The novel anticancer agent nutlin-3 provides a promising alternative for p53 activation by disrupting the interaction between p53 and its negative feedback regulator MDM2. METHODS: We examined whether nutlin-3 enhances apoptosis induction by rhTRAIL and the DR5-selective TRAIL variant D269H/E195R in wild-type p53-expressing ovarian, colon and lung cancer cell lines and in an ex vivo model of human ovarian cancer. RESULTS: Nutlin-3 enhanced p53, p21, MDM2 and DR5 surface expression. Although nutlin-3 did not induce apoptosis, it preferentially enhanced D269H/E195R-induced apoptosis over rhTRAIL. Combination treatment potentiated the cleavage of caspases 8, 9, 3 and PARP. P53 and MDM2 siRNA experiments showed that this enhanced apoptotic effect was mediated by wild-type p53. Indeed, nutlin-3 did not enhance rhTRAIL-induced apoptosis in OVCAR-3 cells harbouring mutant p53. Addition of the chemotherapeutic drug cisplatin to the combination further increased p53 and DR5 levels and rhTRAIL- and D269H/E195R-induced apoptosis. As a proof of concept, we show that the combination of D269H/E195R, nutlin-3 and cisplatin induced massive apoptosis in ex vivo tissue slices of primary human ovarian cancers. CONCLUSION: Nutlin-3 is a potent enhancer of D269H/E195R-induced apoptosis in wild-type p53-expressing cancer cells. Addition of DNA-damaging agents such as cisplatin further enhances DR5-mediated apoptosis.

Inhibition of formyl peptide receptor in high-grade astrocytoma by CHemotaxis Inhibitory Protein of S. aureus.

BACKGROUND: High-grade astrocytomas are malignant brain tumours that infiltrate the surrounding brain tissue and have a poor prognosis. Activation of formyl peptide receptor (FPR1) on the human astrocytoma cell line U87 promotes cell motility, growth and angiogenesis. We therefore investigated the FPR1 inhibitor, Chemotaxis Inhibitory Protein of S. aureus (CHIPS), as a potential anti-astrocytoma drug. METHODS AND RESULTS: FPR1 expression was studied immunohistochemically in astrocytomas WHO grades I-IV. With intracellular calcium mobilisation and migration assays, human ligands were tested for their ability to activate FPR1 on U87 cells and on a cell line derived from primary astrocytoma grade IV patient material. Thereafter, we selectively inhibited these ligand-induced responses of FPR1 with an anti-inflammatory compound called Chemotaxis Inhibitory Protein of S. aureus (CHIPS). U87 xenografts in NOD-SCID mice served to investigate the effects of CHIPS in vivo. FPR1 was expressed in 29 out of 32 (90%) of all grades of astrocytomas. Two human mitochondrial-derived formylated peptides, formyl-methionil-leucine-lysine-isoleucine-valine (fMLKLIV) and formyl-methionil-methionil-tyrosine-alanine-leucine-phenylalanine (fMMYALF), were potent activators of FPR1 on tumour cells. Ligand-induced responses of FPR1-expressing tumour cells could be inhibited with FPR1 inhibitor CHIPS. Treatment of tumour-bearing mice with CHIPS slightly reduced tumour growth and improved survival as compared to non-treated animals (P=0.0019). CONCLUSION: Targeting FPR1 with CHIPS reduces cell motility and tumour cell activation, and prolongs the survival of tumour-bearing mice. This strategy could be explored in future research to improve treatment results for astrocytoma patients.

Expression cloning of a cDNA for the major Fanconi anaemia gene, FAA.

Fanconi anaemia (FA) is an autosomal recessive disorder characterized by a diversity of clinical symptoms including skeletal abnormalities, progressive bone marrow failure and a marked predisposition to cancer. FA cells exhibit chromosomal instability and hyper-responsiveness to the clastogenic and cytotoxic effects of bifunctional alkylating (cross-linking) agents, such as diepoxybutane (DEB) and mitomycin C (MMC). Five complementation groups (A-E) have been distinguished on the basis of somatic cell hybridization experiments, with group FA-A accounting for over 65% of the cases analysed. A cDNA for the group C gene (FAC) was reported and localized to chromosome 9q22.3 (ref.8). Genetic map positions were recently reported for two more FA genes, FAA (16q24.3) and FAD (3p22-26). Here we report the isolation of a cDNA representing the FAA gene, following an expression cloning method similar to the one used to clone the FAC gene. The 5.5-kb cDNA has an open reading frame of 4,368 nucleotides. In contrast to the 63-kD cytosolic protein encoded by the FAC gene, the predicted FAA protein (M(r) 162, 752) contains two overlapping bipartite nuclear localization signals and a partial leucine zipper consensus, which are suggestive of a nuclear localization.

Circulating tumor cells in small-cell lung cancer: a predictive and prognostic factor.

BACKGROUND: Initial response of small-cell lung cancer (SCLC) to chemotherapy is high, and recurrences occur frequently, leading to early death. This study investigated the prognostic value of circulating tumor cells (CTCs) in patients with SCLC and whether changes in CTCs can predict response to chemotherapy. Patients and methods In this multicenter prospective study, blood samples for CTC analysis were obtained from 59 patients with SCLC before, after one cycle, and at the end of chemotherapy. CTCs were measured using CellSearch systems. RESULTS: At baseline, lower numbers of CTCs were observed for 21 patients with limited SCLC (median = 6, range 0-220) compared with 38 patients with extensive stage (median = 63, range 0-14,040). Lack of measurable CTCs (27% of patients) was associated with prolonged survival (HR 3.4; P ≤ 0.001). CTCs decreased after one cycle of chemotherapy; this decrease was not associated with tumor response after four cycles of chemotherapy. CTC count after the first cycle of chemotherapy was the strongest predictor for overall survival (HR 5.7; 95% CI 1.7-18.9; P = 0.004). CONCLUSION: Absolute CTCs after one cycle of chemotherapy in patients with SCLC is the strongest predictor for response on chemotherapy and survival. Patients with low initial CTC numbers lived longer than those with higher CTCs.

Thymidine phosphorylase in cancer cells stimulates human endothelial cell migration and invasion by the secretion of angiogenic factors.

BACKGROUND: Thymidine phosphorylase (TP) is often overexpressed in tumours and has a role in tumour aggressiveness and angiogenesis. Here, we determined whether TP increased tumour invasion and whether TP-expressing cancer cells stimulated angiogenesis. METHODS: Angiogenesis was studied by exposing endothelial cells (HUVECs) to conditioned medium (CM) derived from cancer cells with high (Colo320TP1=CT-CM, RT112/TP=RT-CM) and no TP expression after which migration (wound-healing-assay) and invasion (transwell-assay) were determined. The involvement of several angiogenic factors were examined by RT-PCR, ELISA and blocking antibodies. RESULTS: Tumour invasion was not dependent on intrinsic TP expression. The CT-CM and RT-CM stimulated HUVEC-migration and invasion by about 15 and 40%, respectively. Inhibition by 10 µM TPI and 100 µM L-dR, blocked migration and reduced the invasion by 50-70%. Thymidine phosphorylase activity in HUVECs was increased by CT-CM. Reverse transcription-polymerase chain reaction revealed a higher mRNA expression of bFGF (Colo320TP1), IL-8 (RT112/TP) and TNF-α, but not VEGF. Blocking antibodies targeting these factors decreased the migration and invasion that was induced by the CT-CM and RT-CM, except for IL-8 in CT-CM and bFGF in RT-CM. CONCLUSION: In our cell line panels, TP did not increase the tumour invasion, but stimulated the migration and invasion of HUVECs by two different mechanisms. Hence, TP targeting seems to provide a potential additional strategy in the field of anti-angiogenic therapy.

TRAIL receptor targeting therapies for non-small cell lung cancer: current status and perspectives.

Non-small cell lung cancer (NSCLC) is a common and often fatal malignancy, diagnosed at an advanced stage in more than half of the cases. Chemo-resistance remains a major problem in the treatment of NSCLC patients with conventional chemotherapeutic agents. Therefore main research efforts are focused on the development of novel targeted agents. In this review we provide an overview on the use of TNF-related apoptosis-inducing ligand (TRAIL) receptor targeting agents in NSCLC models and in early clinical studies. Different TRAIL receptor targeting agents are available which have been tested in NSCLC models and some were tested in the clinic. The efficacy of these drugs as single agents in NSCLC models is discussed as well as different mechanisms of resistance that are found in NSCLC cell lines. In order to maximize sensitivity to TRAIL receptor targeting drugs, combined use with other drugs is of interest. The current status of tested combinations of TRAIL receptor targeting agents with other therapeutics, such as classical cytotoxics, Bcl-2 family targeting agents, proteasome inhibitors, EGFR inhibitors, histone deacetylase inhibitors and COX-2 inhibitors as well as their mechanisms in preclinical studies are discussed. Clinical studies on TRAIL targeted therapies in which NSCLC patients were included are discussed and future perspectives are considered.

[Ending up in a wheelchair in a strange way]. / Op onduidelijke wijze in een rolstoel beland.

In this case-report we present a patient with a genetic disease which was first diagnosed in his eighties. The genetic disease is a rare neurologic disease, Kennedy's disease or spinobulbar muscular atrophy (SBMA). We also discuss the genetics of the disease and developments of future therapies.

Synergistic interaction between trifluorothymidine and docetaxel is sequence dependent.

Docetaxel is a microtubule inhibitor that has actions in the S and G(2)-M phase of the cell cycle. The pyrimidine trifluorothymidine (TFT) induces DNA damage and an arrest in the G(2)-M phase. TFT, as part of TAS-102, has been clinically evaluated as an oral chemotherapeutic agent in colon and gastric cancer. The aim of the present study was to determine the optimal administration sequence of TFT and docetaxel and to investigate the underlying mechanism of cytotoxicity. Drug interactions were examined by sulforhodamine B assays and subsequent combination index analyses, and for long-term effects the clonogenic assay was used. A preincubation with docetaxel was synergistic in sulforhodamine B (combination index 0.6-0.8) and clonogenic assays, and was accompanied by a time-dependent cell death induction (17-36%), the occurrence of polynucleation (22%), and mitotic spindle inhibition as determined by flow cytometry and immunostaining. Interestingly, administration of TFT followed by the combination displayed strong antagonistic activity, and was accompanied by less polynucleation and cell death induction than the synergistic combinations. Western blotting showed that the G(2)-M-phase arrest (25-50%) was accompanied by phosphorylation of Chk2 and dephosphorylation of cdc25c in the synergistic combinations. Together, this indicates that synergistic activity requires docetaxel to initiate mitotic failure prior to the activation of TFT damage signaling, whereas antagonism is a result of TFT cell cycle-arrested cells being less susceptible to docetaxel. Caspase 3 activation was low after docetaxel, suggestive of caspase-independent mechanisms of cell death. Taken together, our models indicate that combination treatment with docetaxel and TFT displays strong synergy when docetaxel is given first, thus providing clues for possible clinical studies.

Subcellular localization and nucleocytoplasmic transport of the chromosomal passenger proteins before nuclear envelope breakdown.

As mitosis progresses, the chromosomal passenger proteins (CPPs) Survivin, Aurora B, INCENP and Borealin dynamically colocalize to mitotic structures. Chromosomal passenger proteins are already expressed during G2, whereas the nuclear envelope is only disassembled at the end of prophase. However, the mechanisms that modulate their nucleocytoplasmic localization before nuclear envelope breakdown (NEB) are poorly characterized. Using epitope-tagged proteins, we show that Aurora B, like Survivin, undergoes CRM1-mediated nucleocytoplasmic shuttling, although both proteins lack identifiable 'classical' nuclear transport signals. On the other hand, INCENP resides more stably in the nucleus and contains multiple nuclear localization signals. Finally, Borealin was detected in the nucleolus and the cytoplasm, but its cytoplasmic localization is not directly regulated by CRM1. Coexpression experiments indicate that the nuclear localization of Aurora B, but not of Survivin, is modulated by INCENP and that Survivin prevents the nucleolar accumulation of Borealin. Our data reveal that, in contrast to their closely related localization during mitosis, the nucleocytoplasmic localization of the CPPs before NEB is largely unrelated. Furthermore, the specific effect of INCENP and Survivin on the localization of Aurora B and Borealin, respectively, suggests that different complexes of CPPs may exist not only during mitosis, as recently reported, but also before NEB.

Paclitaxel and vincristine potentiate adenoviral oncolysis that is associated with cell cycle and apoptosis modulation, whereas they differentially affect the viral life cycle in non-small-cell lung cancer cells.

Chemotherapy, including microtubule (MT)-interacting agents, can enhance the tumor-eradicating activity of replication-competent adenoviruses. The purpose of this study was to obtain more insight into the mechanism underlying this enhancement that may be exploited for the development of improved therapy. Two MT-interacting agents with opposite activity, paclitaxel (PTX) that stabilizes and vincristine (VCR) that destabilizes MTs, were found to synergistically enhance adenoviral oncolysis in non-small-cell lung cancer (NSCLC) cells. To explore the possibility that these drugs affect the viral life cycle by modulating adenoviral gene expression, we used a quantitative reverse transcription-polymerase chain reaction assay and found that PTX, but not VCR, increased the expression of E1A13S, ADP and Penton genes, which correlated with an increase in viral particle assembly and release. Next, the effect of combined treatment on cell-cycle progression was studied. Both drugs suppressed adenovirus-induced S-phase arrest and instead caused G2/M arrest, which was accompanied by an increase in apoptotic cells. Taken together, the enhancement of oncolysis by MT-interacting drugs appears not to require specific MT transport or scaffold functions. Our findings suggest that MT-interacting drug-induced cellular signals that modulate cell-cycle arrest and apoptosis are primarily on the basis of their oncolysis-enhancing activity.

Involvement of the Fanconi's anemia protein FAC in a pathway that signals to the cyclin B/cdc2 kinase.

Lymphoblastoid cell lines derived from patients with the chromosomal instability disorder Fanconi's anemia (FA) are hyperresponsive to G2 delay and apoptosis induced by cross-linking agents such as mitomycin C (MMC). Here, we investigated whether the protein defective in FA complementation group C (FA-C) cells functions in a pathway that signals to the cdc2 kinase complex, which controls mitotic progression. FA-C lymphoblasts treated with a low dose of MMC (1-5 microM, 1 h) exhibited a protracted G2-M arrest and subsequent apoptosis by 2 days after treatment. This G2-M arrest was mediated by persistent inactivation of the cyclin B1/cdc2 kinase complex characterized by both sustained accumulation of cyclin B1 and tyrosine phosphorylation of cdc2. In phenotypically corrected (wild-type) cells, the same treatment induced only temporal G2-M arrest, associated with a transient inactivation of the cyclin B1/cdc2 kinase complex, after which cells resumed cycling. Treatment with higher dosages (15-30 microM, 1 h) resulted in S-phase arrest and induced a similar high level of apoptosis in FA-C and wild-type cells, accompanied by degradation of cyclin B1 and dephosphorylation of cdc2. In low-dose treated G2-M-arrested FA-C cells, caffeine-dependent activation of cdc2 released the G2-M block but failed to protect against apoptosis, suggesting that apoptosis was not a direct consequence of persistent cdc2 kinase inactivation. Thus, at low doses of MMC, FA-C cells exhibit a unique cyclin B1/cdc2 response that is not observed in wild-type cells treated with an equitoxic high dosage of cross-linker. Although these results do not necessarily implicate a role for FAC in regulating cyclin B/cdc2 kinase activity, available evidence suggests that the FAC protein is involved in a cross-link damage avoidance pathway that signals to this kinase complex.

Chemotherapy triggers apoptosis in a caspase-8-dependent and mitochondria-controlled manner in the non-small cell lung cancer cell line NCI-H460.

Chemotherapy-induced apoptosis is generally thought to be dependent on a pathway headed by caspase-9. This model is primarily based on studies performed in leukemia cells; however, little is known about caspase cascades in relatively resistant solid tumor cells, including non-small cell lung cancer (NSCLC) cells. Using the NSCLC cell line NCI-H460 (H460), here, we studied the effect of stable expression of various caspase inhibitors on apoptosis induced by the anticancer drugs cisplatin, topotecan, and gemcitabine. Interestingly, overexpression of caspase-9S and X-linked inhibitor of apoptosis (XIAP), both able to inhibit caspase-9 activity, failed to block apoptosis. In contrast, stable expression of caspase-8 inhibitors, such as cytokine response modifier A (CrmA) and dominant-negative caspase-8, almost completely abrogated apoptosis and also enhanced clonogenic survival. Caspase-8 activation in H460 cells was not mediated by death receptors, inasmuch as overexpression of dominant-negative Fas-associated death domain (FADD-DN) did not prevent procaspase-8 cleavage and subsequent apoptosis. However, stable expression of Bcl-2 and Bcl-xL did suppress these apoptotic events, including the release of cytochrome c from mitochondria, which was observed in drug-treated H460 cells. In the NSCLC cell line H460, we, thus, provide evidence for the existence of a novel drug-inducible apoptotic pathway in which activation of caspase-8, and not of caspase-9, forms the apical and mitochondria-dependent step that subsequently activates the downstream caspases.

Transcriptional regulation of retinoic acid receptor beta in retinoic acid-sensitive and -resistant P19 embryocarcinoma cells.

As in other embryocarcinoma (EC) cell lines retinoic acid (RA) rapidly induces expression of the nuclear retinoic acid receptor (RAR) beta in murine P19 EC cells, while RAR alpha is expressed constitutively. In the RA-resistant P19 EC-derived RAC65 cells, however, there is no such induction and an aberrant (smaller) RAR alpha transcript is expressed. RAR gamma 1 is expressed at low levels in both cell lines. To study the regulation of the RAR beta gene and the possible involvement of RAR alpha protein in transcriptional activation of the RAR beta gene we transfected these cells with a construct containing a 1.6 kb promoter fragment of the human RAR beta gene fused to the CAT gene. Upon transient assays in P19 EC cells CAT activity is enhanced rapidly by RA, to more than 100-fold in a concentration-dependent fashion. On the contrary no activity can be observed in the RA-resistant RAC65 cells; however, co-transfection of hRAR alpha, hRAR beta or hRAR gamma 1 restores the RA-dependent induction of CAT activity. These results clearly show that RAR alpha and RAR gamma 1 can transactivate the RAR beta gene; that RAR beta can stimulate its own expression and that resistance to RA in RAC65 cells is probably due to the altered RAR alpha transcript present in these cells.

Aggregation and cell cycle dependent retinoic acid receptor mRNA expression in P19 embryonal carcinoma cells.

Differentiation of P19 EC cells along different pathways into derivatives resembling cells of the three embryonic germ layers is accompanied by characteristic differences in modulation of expression of each of the three retinoic acid receptor genes, RAR alpha, -beta and -gamma. Differentiation induced by addition of RA to P19 EC cells cultured in monolayer is accompanied by a rapid increase in expression of both RAR alpha and -beta. Induction of RAR beta occurs in a characteristic biphasic manner, suggesting that multiple factors and/or different mechanisms are involved in controlling its expression. RAR beta mRNA is induced to a far higher level during early aggregation in the presence of RA than during early differentiation in monolayer, suggesting that the direction of differentiation depends on the number and/or ratio of alpha and beta type of RA receptors. Aggregation of P19 EC cells in the presence of RA, but not DMSO, is accompanied by repression of RAR gamma, suggesting that the expression of RAR beta and RAR gamma during neuroectodermal differentiation is mutually exclusive. The effects of RA on RAR expression are significantly greater in G1 than in S-phase of the cell cycle. These results extend previous observations that commitment to differentiation is cell cycle dependent and indicates that critical target gene regulation in response to RA has to take place in G1 for differentiation to occur.

Expression of X-linked inhibitor of apoptosis as a novel prognostic marker in radically resected non-small cell lung cancer patients.

PURPOSE: To assess the pattern of expression and the prognostic value of the inhibitor of apoptosis family member X-linked inhibitor of apoptosis (XIAP; MIHA/ILP-a) in radically resected non-small cell lung cancer patients. EXPERIMENTAL DESIGN: The expression of XIAP and its relationship with overall survival was analyzed by immunohistochemistry on tumors from 144 patients with early-stage non-small cell lung cancer. In addition, the apoptotic and mitotic index, Ki-67, p53, and bcl-2 levels were also assessed. RESULTS: XIAP expression was specific for tumor cells, and the pattern was cytoplasmic. The median expression of XIAP was 20%, and when this value was used as a cutoff point for statistical analyses, 63 of the samples were considered high XIAP-expressing and 81 low XIAP-expressing. Surprisingly, high XIAP-expressing patients had a longer overall survival than the group expressing lower levels (60 versus 24 months of median survival; log rank, P = 0.01). The positive impact of XIAP expression on survival was confirmed by multivariate analysis (P = 0.026). Although no correlation was observed between XIAP expression and the apoptotic index, a significant inverse correlation was observed between XIAP, Ki-67 (P = 0.006), and mitotic index (P = 0.04). CONCLUSIONS: The unexpected inverse correlation of XIAP with proliferation markers and the absence of correlation with apoptotic index, coupled with its role as an independent positive prognostic factor for survival in radically resected NSCLC patients imply a more complex role for XIAP in tumor biology than anticipated by in vitro data.

E1A functions as a coactivator of retinoic acid-dependent retinoic acid receptor-beta 2 promoter activation.

The retinoic acid (RA) receptor (RAR) beta 2 promoter is strongly activated by RA in embryonal carcinoma (EC) cells. We examined this activation in the P19 EC-derived END-2 cell line and in E1A-expressing counterparts and found strong RA-dependent RAR beta 2 promoter activation in the E1A-expressing cells, which was not observed in the parental cell line, indicating a possible role for E1A in RAR beta 2 activation. In transient transfection assays, E1A functioned as a coactivator of RA-dependent RAR beta 2 promoter activation and, moreover, was able to restore this activation in cells lacking RAR beta 2 activation. By deletion analysis, two regions in the RAR beta 2 promoter were identified that mediate the stimulatory effect of E1A: the RA response element and TATA box-containing region and a more up-stream region between -180 and -63, in which a cAMP response element-related motif was identified as a target element for E1A. In addition, determination of endogenous E1A-like activity by measuring E2A promoter activity in transient transfection assays in EC and differentiated cells revealed a correlation between RA-dependent RAR beta 2 promoter activation and the presence of this activity, suggesting an important role for the cellular equivalent of E1A in regulation of the RAR beta 2 promoter.