SLC28A3 genotype and gemcitabine rate of infusion affect dFdCTP metabolite disposition in patients with solid tumours.

BACKGROUND: Gemcitabine is used for the treatment of several solid tumours and exhibits high inter-individual pharmacokinetic variability. In this study, we explore possible predictive covariates on drug and metabolite disposition. METHODS: Forty patients were enrolled. Gemcitabine and dFdU concentrations in the plasma and dFdCTP concentrations in peripheral blood mononuclear cell were measured to 72 h post infusion, and pharmacokinetic parameters were estimated by nonlinear mixed-effects modelling. Patient-specific covariates were tested in model development. RESULTS: The pharmacokinetics of gemcitabine was best described by a two-compartment model with body surface area, age and NT5C2 genotype as significant covariates. The pharmacokinetics of dFdU and dFdCTP were adequately described by three-compartment models. Creatinine clearance and cytidine deaminase genotype were significant covariates for dFdU pharmacokinetics. Rate of infusion of <25 mg m(-2) min(-1) and the presence of homozygous major allele for SLC28A3 (CC genotype) were each associated with an almost two-fold increase in the formation clearance of dFdCTP. CONCLUSION: Prolonged dFdCTP systemic exposures (≥72 h) were commonly observed. Infusion rate <25 mg m(-2) min(-1) and carriers for SLC28A3 variant were each associated with about two-fold higher dFdCTP formation clearance. The impacts of these covariates on treatment-related toxicity in more selected patient populations (that is, first-line treatment, single disease state and so on) are not yet clear.

Cross-validation study of class III beta-tubulin as a predictive marker for benefit from adjuvant chemotherapy in resected non-small-cell lung cancer: analysis of four randomized trials.

BACKGROUND: The IALT, JBR.10, ANITA and Cancer and Leukemia Group B 9633 trials compared adjuvant chemotherapy with observation for patients with resected non-small-cell lung cancer (R-NSCLC). Data from the metastatic setting suggest high tumor class III beta-tubulin (TUBB3) expression is a determinant of insensitivity to tubulin-targeting agents (e.g. vinorelbine, paclitaxel). In 265 patients from JBR.10 (vinorelbine-cisplatin versus observation), high TUBB3 was an adverse prognostic factor and was associated (nonsignificantly) with 'greater' survival benefit from chemotherapy. We explored this further in additional patients from JBR.10 and the other three trials. PATIENTS AND METHODS: TUBB3 immunohistochemical staining was scored for 1149 patients on the four trials. The original JBR.10 cut-off scores were used to classify tumors as TUBB3 high or low. The prognostic and predictive value of TUBB3 on disease-free survival (DFS) and overall survival (OS) was assessed by Cox models stratified by trial and adjusted for clinical factors. RESULTS: High TUBB3 expression was prognostic for OS [hazard ratio (HR)=1.27 (1.07-1.51), P=0.008) and DFS [HR=1.30 (1.11-1.53), P=0.001). TUBB3 was not predictive of a differential treatment effect [interaction P=0.20 (OS), P=0.23 (DFS)]. Subset analysis (n=420) on vinorelbine-cisplatin gave similar results. CONCLUSIONS: The prognostic effect of high TUBB3 expression in patients with R-NSCLC has been validated. We were unable to confirm a predictive effect for TUBB3.

Design and modification of EGF4KDEL 7Mut, a novel bispecific ligand-directed toxin, with decreased immunogenicity and potent anti-mesothelioma activity.

BACKGROUND: Potency, immunogenicity, and toxicity are three problems that limit the use of targeted toxins in solid tumour therapy. METHODS: To address potency, we used genetic engineering to develop a novel bispecific ligand-directed toxin (BLT) called EGF4KDEL, a novel recombinant anti-mesothelioma agent created by linking human epidermal growth factor (EGF) and interleukin-4 (IL-4) to truncated pseudomonas exotoxin (PE38) on the same single-chain molecule. Immunogenicity was reduced by mutating seven immunodominant B-cell epitopes on the PE38 molecule to create a new agent, EGF4KDEL 7Mut. RESULTS: In vitro, bispecific EGF4KDEL showed superior anti-mesothelioma activity compared with its monospecific counterparts. Toxicity in mice was diminished by having both ligands on the same molecule, allowing administration of a 10-fold greater dose of BLT than a mixture of monomeric IL4KDEL and EGFKDEL. EGF4KDEL 7Mut, retained all of its functional activity and induced about 87% fewer anti-toxin antibodies than mice given the parental, non-mutated form. In vivo, intraperitoneal (IP) injection of the BLT showed significant (P<0.01) and impressive effects against two aggressive, malignant IP mesothelioma models when treatment was begun 14-16 days post tumour innoculation. CONCLUSION: These data show that EGF4KDEL 7Mut is a promising new anti-mesothelioma agent that was developed to specifically address the obstacles facing clinical utility of targeted toxins.

Phase I study of bortezomib and cetuximab in patients with solid tumours expressing epidermal growth factor receptor.

Bortezomib inhibits nuclear factor-kappaB (NF-kappaB). Cetuximab is a chimeric mouse-human antibody targeted against epidermal growth factor receptor (EGFR). We hypothesised that concomitant blockade of NF-kappaB and EGFR signalling would overcome EGFR-mediated resistance to single-agent bortezomib and induce apoptosis through two molecular pathways. The aim of this phase I trial was to establish the maximum tolerated dose (MTD) for bortezomib plus cetuximab in patients with EGFR-expressing epithelial tumours. The 21-day treatment cycle consisted of bortezomib administered on days 1 and 8 through dose escalation (1.3-2 mg m(-2)). Cetuximab was delivered at a dose of 250 mg m(-2) on days 1, 8 and 15 (400 mg m(-2) day 1 cycle 1). A total of 37 patients were enroled and given a total 91 cycles. No grade > or =3 haematological toxicity was noted. Non-hematological grade > or =3 toxicities included fatigue (22% of patients), dyspnoea (16%) and infection (11%). The MTD was not reached at the highest tested bortezomib dose (2.0 mg m(-2)). Efficacy outcomes included disease progression in 21 patients (56.7%) and stable disease (SD) at 6 weeks in 16 patients (43.3%). Five of the six patients with SD at 12 weeks were diagnosed with cancers of the lungs or head and neck. This combination therapy was moderately effective in extensively pretreated patients with non-small cell lung or head and neck cancers and warrants further investigation.

Activated 4E-BP1 represses tumourigenesis and IGF-I-mediated activation of the eIF4F complex in mesothelioma.

BACKGROUND: Insulin-like growth factor (IGF)-I signalling stimulates proliferation, survival, and invasion in malignant mesothelioma and other tumour types. Studies have found that tumourigenesis is linked to dysregulation of cap-dependent protein translation. METHODS: The effect of IGF stimulation on cap-mediated translation activation in mesothelioma cell lines was studied using binding assays to a synthetic 7-methyl GTP-cap analogue. In addition, cap-mediated translation was genetically repressed in these cells with a dominant active motive of 4E-BP1. RESULTS: In most mesothelioma cell lines, IGF-I stimulation resulted in a hyperphosphorylation-mediated inactivation of 4E-BP1 compared with that in normal mesothelial cells. An inhibitor of Akt diminished IGF-I-mediated phosphorylation of 4E-BP1, whereas inhibiting MAPK signalling had no such effect. IGF-I stimulation resulted in the activation of the cap-mediated translation complex as indicated by an increased eIF4G/eIF4E ratio in cap-affinity assays. Akt inhibition reversed the eIF4G/eIF4E ratio. Mesothelioma cells transfected with an activated 4E-BP1 protein (4E-BP1(A37/A46)) were resistant to IGF-I-mediated growth, motility, and colony formation. In a murine xenograft model, mesothelioma cells expressing the dominant active 4E-BP1(A37/A46) repressor protein showed abrogated tumourigenicity compared with control tumours. CONCLUSION: IGF-I signalling in mesothelioma cells drives cell proliferation, motility, and tumourigenesis through its ability to activate cap-mediated protein translation complex through PI3K/Akt/mTOR signalling.

Differential specificity for binding of retinoblastoma binding protein 2 to RB, p107, and TATA-binding protein.

The growth suppressor activities of the RB and p107 products are believed to be mediated by the reversible binding of a heterogeneous family of cellular proteins to a conserved T/E1A pocket domain that is present within both proteins. To study the functional role of these interactions, we examined the properties of cellular retinoblastoma binding protein 2 (RBP2) binding to RB, p107, and the related TATA-binding protein (TBP) product. We observed that although RBP2 bound exclusively to the T/E1A pocket of p107, it could interact with RB through independent T/E1A and non-T/E1A domains and with TBP only through the non-T/E1A domain. Consistent with this observation, we found that a mutation within the Leu-X-Cys-X-Glu motif of RBP2 resulted in loss of ability to precipitate p107, while RB- and TBP-binding activities were retained. We located the non-T/E1A binding site of RBP2 on a 15-kDa fragment that is independent from the Leu-X-Cys-X-Glu motif and encodes binding activity for RB and TBP but does not interact with p107. Despite the presence of a non-T/E1A binding site, however, recombinant RBP2 retained the ability to preferentially precipitate active hypophosphorylated RB from whole-cell lysates. In addition, we found that cotransfection of RBP2 can reverse in vivo RB-mediated suppression of E2F activity. These findings confirm the differential binding specificities of the related RB, p107, and TBP proteins and support the presence of multifunctional domains on the nuclear RBP2 product which may allow complex interactions with the cellular transcription machinery.

Immunohistochemical analysis of the p16INK4 cyclin-dependent kinase inhibitor in malignant mesothelioma.

BACKGROUND: The identification in 1994 of the CDKN2 gene as a target for mutations in a wide range of human cancers, including malignant mesothelioma, has been controversial because subsequent studies have detected a lower frequency of CDKN2 gene mutations in primary tumors than in cultured cell lines. These reports raised the hypothesis that another gene, distinct from CDKN2, might be the target of the chromosome 9p21 deletions frequently observed in these tumors. PURPOSE: To address whether inactivation of CDKN2 function is an essential event in the etiology of malignant mesothelioma, we examined p16INK4 protein expression in primary thoracic mesotheliomas, in nonmalignant pleural tissues, and in independent mesothelioma cell lines. We also studied the growth rate of tumor cell lines following stable transfection of CDKN2 gene. METHODS: Retinoblastoma (Rb) and p16INK4 protein expression was determined by immunohistochemical analysis from archival paraffin specimens of 12 primary thoracic mesotheliomas and a nonmalignant pleural biopsy specimen. In addition, protein immunoblot analysis for Rb and p16INK4 expression was conducted on 15 independent mesothelioma cell lines, and the ability of a transfected CDKN2 gene to suppress the growth of the mesothelioma cell lines H2373 and H2461 in vitro was examined. RESULTS: We demonstrated abnormal p16INK4 expression in 12 of 12 primary mesothelioma specimens and in 15 of 15 mesothelioma cell lines. All tumor specimens and the tumor cell lines showed expression of wild-type Rb protein. In addition, we have confirmed the ability of a transfected CDKN2 gene to suppress growth of two independent mesothelioma cell lines. CONCLUSIONS: Immunohistochemical analysis of the p16INK4 gene product is feasible in archival biopsy samples. With this analysis, CDKN2 gene inactivation can be determined in tumors that are contaminated with nonmalignant cells. Furthermore, since loss of p16INK4 protein expression can result from both genetic (gene mutations) and epigenetic (abnormal DNA hypermethylation) mechanisms, as we and others have shown recently, examination of protein expression is a highly sensitive method for analyzing the CDKN2 status in large numbers of tumor samples. IMPLICATIONS: This study suggests that inactivation of the CDKN2 gene is an essential step in the etiology of malignant mesotheliomas. Defining the role of the p16INK4:Rb tumor suppressor pathway and its immediate downstream substrates will be an important goal in designing future therapeutic strategies.

Immunohistochemical detection of the cyclin-dependent kinase inhibitor 2/multiple tumor suppressor gene 1 (CDKN2/MTS1) product p16INK4A in archival human solid tumors: correlation with retinoblastoma protein expression.

The retinoblastoma (RB) and cyclin-dependent kinase inhibitor 2/multiple tumor suppressor gene 1 (CDKN2/MTS1) tumor suppressor genes play important roles in the regulation of the cell cycle. The protein products of these two genes, pRB and p16INK4A ("p16"), respectively, inhibit progression from G1 to S phase. Moreover, p16 has been shown to exert its function through inhibition of CDK4-mediated phosphorylation of pRB. Both genes have been found to be mutated or deleted in a wide range of primary human tumors and tumor cell lines. However, the presence of CDKN2/MTS1 containing nonneoplastic elements in every tumor specimen may contribute to the apparent lower deletion detection rate in resected neoplasms compared to cell lines. We have developed an immunohistochemical assay that allows us to assess p16 expression in formalin-fixed, paraffin-embedded tissues. As controls, we used paraffin-embedded pellets of cell lines with well-defined p16 status (four positive and four negative lines), as well as routinely processed nude mouse xenografts of two p16-positive cell lines. p16-negative cells were characterized by the absence of nuclear staining, whereas cytoplasmic staining was variable. In neoplastic and normal tissues, the level of p16 generally appeared to be low. We tested 75 random human malignancies from 4 different anatomic sites for p16 expression and correlated the findings with the immunohistochemical presence or absence of pRB. Twenty % of tumors selectively lacked pRB, while 37% of neoplasms had undetectable p16. In 43% of all carcinomas, both pRB and p16 could be detected. Significant differences existed in the expression of both tumor suppressor genes between carcinomas from different sites. Breast cancers had the highest rate of p16 negativity (13 of 20). Our data show that: (a) immunohistochemistry may be a suitable modality to screen for RB and CDKN2/MTS1 abnormalities in paraffin-embedded tissues; (b) undetectable levels of p16 expression occur at a relatively high frequency; (c) p16 and pRB expression in common human malignancies is not mutually exclusive; (d) loss of function of both tumor suppressor genes appears to be a distinctly uncommon phenomenon; and (e) different types of carcinomas have variable rates of disturbance in the p16/pRB pathway.

Rb and p16INK4a expression in resected non-small cell lung tumors.

Inactivation of the cyclin-dependent kinase inhibitor p16INK4a (CDKN2/MTS1) is documented in a wide variety of cancer cell lines and tumors. We have shown that loss of p16INK4a protein expression is a common event in early stage non-small cell lung cancer (NSCLC), correlates with a significantly worse survival, and is more common in higher stage disease. One hundred NSCLC tumors from patients undergoing definitive thoracotomies at a single institution were examined for p16INK4a and retinoblastoma protein (pRB) expression. Abnormal pRB staining was identified in 15% of the tumors, whereas 51% possessed aberrant p16INK4a protein expression. Tumors with aberrant expression of p16INK4a by immunohistochemistry were associated with a significantly worse survival (P=0.04). Additionally, the inverse correlation of pRB and p16INK4a expression previously noted in lung cancer cell lines and tumors was confirmed in this large cohort of patients, with 65% of the tumors demonstrating inverse expression of pRB and p16INK4a (p=0.00019). A statistically significant increase in aberrant p16INK4a expression, as well as inverse expression of p16INK4a and pRB, was seen with increasing pathological stage of disease. These findings establish the prognostic significance (of the absence of p16INK4, in resected NSCLC and confirm the critical importance of disrupting the pathway of cyclin-dependent kinase-mediated phosphorylation of pRB in the molecular oncogenesis and progression of NSCLC.

Human lung carcinomas express Fas ligand.

To reach a clinically detectable size, neoplasms must be able to suppress or evade a host immune response. Activated T cells may enter apoptosis in the presence of Fas ligand (FasL) (1), and tissue expression of FasL has been shown to contribute to immune privilege in the eye and testis (2, 3). We have demonstrated that all human lung carcinoma cell lines tested (16 of 16) express a Mr 38,000 protein consistent with FasL by immunoblotting, whereas the majority of resected tumors (23 of 28) show positive staining for FasL by immunohistochemistry. DNA sequencing of reverse transcription-PCR products from lung cancer cells and resected lung tumors confirms the presence of human FasL mRNA in these neoplastic tissues. Furthermore, lung carcinoma cells are capable of killing a Fas-sensitive human T cell line (Jurkat) in coculture experiments; this killing was inhibited by a recombinant form of the soluble portion of the Fas receptor (FasFc). FasL expression by neoplastic cells represents a potential mechanism for peripheral deletion of tumor-reactive T-cell clones.

Alternative splicing of the RBP1 gene clusters in an internal exon that encodes potential phosphorylation sites.

We have isolated cDNA and genomic clones for the human retinoblastoma binding protein 1 (RBP1) gene, and have identified alternative splicing of RBP1 clustered within a 207-nucleotide internal exon. Three of the predicted RPB1 peptides share amino-terminal and carboxy-terminal domains, while a fourth species encodes a distinct carboxy-terminal domain. Functional analysis of these peptides demonstrated that they are capable of precipitating retinoblastoma (RB) protein in vitro from K562 cell lysates, but cannot bind to mutant RB protein. However, each of the RBP1 peptides differed within an internal exon that contains potential casein kinase II and p34cdc2 phosphorylation sites. Immunoblot analysis using polyclonal alpha-RBP1 antiserum revealed that the RBP1 protein is expressed in a wide range of cell lines of differing histologic type and migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis predominantly as a 200-kDa protein. Immunohistochemical analysis using the alpha-RBP1 antiserum demonstrated a distinct nuclear staining pattern that was eliminated when the antiserum was preabsorbed with RBP1 peptide. The RBP1 gene encodes a widely expressed 200-kDa nuclear protein and undergoes alternative splicing that predicts a family of RB-binding peptides.

Absence of p16INK4 protein is restricted to the subset of lung cancer lines that retains wildtype RB.

Cell cycle dependent phosphorylation of the RB tumor suppressor protein is mediated by a family of G1 cyclin dependent kinases (cdks) and cyclins including the activated cdk4:cyclin D complex. The identification of a cdk4 inhibitor, p16INK4, as a target for mutations in cultured tumor lines and primary tumors suggested that RB activity may be affected in these cells. We have examined 88 lung cancer lines for p16INK4 protein expression and have observed a striking inverse correlation between the presence of p16INK4 and wildtype RB. We demonstrated that only 6/55 (11%) of small cell lung cancer (SCLC) samples had absent p16INK4 protein, and all 6 belonged to the rare subset of SCLC with wildtype RB expression. Conversely of 48 SCLC samples with absent or mutant RB, all showed detectable levels of p16INK4 protein. In contrast, we observed that 23/33 (70%) of non-SCLC samples had loss of p16INK4. Twenty-two of 26 non-SCLC lines with wildtype RB had absent p16INK4 while 6 of 7 non-SCLC lines with absent or mutant RB had detectable p16INK4. The inverse correlation of RB and p16INK4 expression and the absence of p16INK4 inactivation in RB (-/-) SCLC lines (0/48) confirms a common p16INK4/RB growth suppressor pathway in human cancers and provides evidence that p16INK4, and not an adjacent gene on chromosome 9p, is a specific target for mutational events.

Re-expression of p16INK4a in mesothelioma cells results in cell cycle arrest, cell death, tumor suppression and tumor regression.

Absence of expression of the p16IKN4a gene product is commonly observed in mesothelioma tumors and cell lines, while wild-type pRB expression is maintained. We have examined the biologic and potential therapeutic role of re-expressing p16INK4a gene product in mesothelioma cells and tumors. Following transduction with a p16INK4a expressing adenovirus (Adp16), over-expression of p16INK4a in mesothelioma cells resulted in cell cycle arrest, inhibition of pRB phosphorylation, diminished cell growth, and eventual death of the transduced cells. Expression of p16INK4a protein was accompanied by decreased expression of pRB as detected by immunoblot and immunohistochemistry. Experiments in mesothelioma xenografts demonstrated inhibition of tumor formation, tumor growth arrest and diminished tumor size and spread. p16INK4a gene product expression was also demonstrated in intraperitoneal xenografts of human mesothelioma cells. These results demonstrate that p16INK4a gene transfer may play a therapeutic role in the treatment of mesothelioma.

RB protein status and clinical correlation from 171 cell lines representing lung cancer, extrapulmonary small cell carcinoma, and mesothelioma.

We have studied RB protein expression in 171 cell lines derived from patients with small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), pulmonary carcinoid, mesothelioma, and extrapulmonary small cell cancer (EPSC) and have correlated this data with clinical outcome. We detected absent or aberrant RB protein expression in 66/75 SCLC, 12/80 NSCLC, 1/6 carcinoid, 0/5 mesothelioma, and 4/5 EPSC samples. In addition, we observed integration of human papilloma virus (HPV) DNA in the single EPSC cell line that retained wildtype RB protein. We did not detect integration of HPV, SV40 or adenoviral DNA in other tumor samples with wildtype RB status. We also noted a stable, hypophosphorylated mutant RB in 12 SCLC and 3 NSCLC samples which might have been falsely interpreted as wildtype by current immunohistochemical techniques. Analysis of the matched clinical data showed no associations between RB status and age, sex, extent of disease, performance status, smoking history, and previous treatment. In addition, retrospective analyses showed no consistent correlation of RB protein expression with either best clinical response, overall survival, or in vitro chemotherapeutic drug sensitivity. The stable expression of RB after gene transfection into RB(-) SCLC cells, however, resulted in a trend toward increased in vitro resistance to etoposide, cisplatin and doxorubicin.

Partial inactivation of the RB product in a family with incomplete penetrance of familial retinoblastoma and benign retinal tumors.

While familial retinoblastoma has served as the paradigm for the two-hit theory of tumorigenesis and for the concept of the tumor suppressor gene, the etiology of incomplete penetrance of familial retinoblastoma is poorly understood. To address the molecular basis for this phenotype we have studied the functional properties of a mutant Rb gene identified in a kindred with incomplete penetrance of familial retinoblastoma and evidence for regressed retinal lesions (retinomas). In contrast to all previously isolated RB mutant proteins, we demonstrated that the mutant product from this kindred retained the wildtype properties of nuclear localization, the ability to undergo hyperphosphorylation in vivo, and the capacity to suppress growth of RB(-) cells. Protein binding ('pocket') activity, however, was defective defining a new class of RB mutant with partial inactivation. The presence of this unique RB mutant in the germline of obligate carriers with incomplete penetrance and regressed retinal lesions suggests a molecular basis for this phenotype and supports the hypothesis that a minimum 'RB threshold' level of protein binding activity is required to suppress tumorigenesis.

Fas ligand expression in the bone marrow in myelodysplastic syndromes correlates with FAB subtype and anemia, and predicts survival.

Increased apoptosis in the bone marrow (BM) may contribute to the cytopenias that occur in myelodysplastic syndromes (MDS). The Fas receptor, Fas ligand (FasL) pathway is a major mechanism of apoptosis. Since hematopoietic progenitors can express the Fas receptor, they may be susceptible to apoptosis induced by FasL-expressing cells. We examined FasL expression in the BM of patients with MDS (n = 50), de novo acute myeloid leukemia (AML; n = 10), AML following prior MDS (n = 6), and normal controls (n = 6). Compared to controls, FasL expression was increased in MDS, and was highest in AML. In MDS, FasL expression was seen in myeloid blasts, erythroblasts, maturing myeloid cells, megakaryocytes and dysplastic cells, whereas in AML, intense expression was seen in the blasts. FasL expression correlated with the FAB subtype groups of MDS, and also correlated directly with the percentage of abnormal metaphases on cytogenetic analysis. The FasL expressed in MDS BM inhibited the growth of clonogenic hematopoietic progenitors. This inhibition could be blocked by a soluble recombinant FasFc protein. In MDS, FasL expression in the initial diagnostic BM was higher in patients who were more anemic, correlated directly with red cell transfusion requirements over the subsequent course of the disease, and was predictive of survival. These studies indicate that FasL expression in MDS is of prognostic significance, and suggest that pharmacological blockade of the Fas-FasL pathway may be of clinical benefit.

Adenovirus-mediated delivery of p16 to p16-deficient human bladder cancer cells confers chemoresistance to cisplatin and paclitaxel.

We have previously established the efficacy of adenoviral gene delivery vectors for the treatment of bladder carcinoma in vivo. In the present work, we developed a gene therapy strategy for bladder cancer based on the replacement of the tumor suppressor p16, which is known to be mutated or deleted in a variety of human tumors, including those derived from the bladder. Previous reports have demonstrated that reconstitution of p16 has marked effects on the proliferative capacity of tumor cell lines both in vitro and in vivo, and that p16 expression causes resistance to some chemotherapeutic agents. In the present study, we describe the construction of the recombinant adenovirus Adp16, expressing the p16 gene, to evaluate the effects of transient p16 replacement in the context of bladder carcinoma. To identify candidate target cell lines, we screened a panel of bladder cancer cell lines for p16 and retinoblastoma (RB) protein expression. We demonstrate that Adp16 can mediate high-efficiency p16 replacement to the p16-negative cell lines EJ and UMUC-3. In addition, the reconstitution of p16 to the highly transducible p16-negative, RB-positive bladder cancer cell line EJ caused a profound inhibition of cell proliferation mediated by arrest in the G1 phase of the cell cycle. In contrast, the p16-positive, RB-negative cell line J82 was unaffected by this treatment. However, when adenovirally mediated p16 replacement was combined with the chemotherapeutics cisplatin and paclitaxel, a marked chemoresistance was observed in genetically corrected cells. This work has implications for future gene therapy strategies based on p16 replacement.

Fas ligand is highly expressed in acute leukemia and during the transformation of chronic myeloid leukemia to blast crisis.

Fas ligand (FasL) induces apoptosis in susceptible Fas-bearing cells and is critically involved in regulating T-cell immune responses. It is highly expressed in several human malignancies, and a role in the suppression of antitumor immune responses has been suggested. We evaluated FasL expression in leukemia and normal hematopoietic cells. By Western blotting, all acute leukemic cell lines (n = 9) and primary samples of acute leukemic marrow (n = 4) revealed high levels of FasL. In contrast, much weaker signals were observed in samples of normal marrow (n = 5), and either weak or intermediate expression was seen in chronic myeloid leukemia (CML) in chronic phase (n = 7). Additional leukemic samples were examined by immunohistochemistry. Staining for FasL was negative in 7 of 9 cases of chronic-phase CML, whereas all cases of CML in blast crisis (n = 6), acute lymphoblastic leukemia (n = 6), and acute myeloid leukemia (n = 11) stained strongly in 60 to 100% of nucleated cells. FasL+ leukemic cell lines did not trigger Fas-mediated apoptosis in either Jurkat cells or activated human T lymphocytes, possibly related to the intracellular location of the ligand. Western analysis of normal marrow subpopulations revealed that most FasL in marrow mononuclear cells was expressed by CD7+ lymphocytes. FasL also was strongly expressed in CD34+ hematopoietic progenitor cells from both normal and chronic-phase CML marrow, suggesting a correlation with primitive maturation stage. In summary, high levels of FasL expression were associated with aggressive biologic behavior in leukemia, including transformation of CML to blast crisis. This could potentially represent a response to loss of proapoptotic Fas signaling, which is known to occur in acute leukemic blasts.

Antisense oligonucleotide targeting eukaryotic translation initiation factor 4E reduces growth and enhances chemosensitivity of non-small-cell lung cancer cells.

Elevated levels of eukaryotic translation initiation factor 4E (eIF4E) enhance translation of many malignancy-related proteins, such as vascular endothelial growth factor (VEGF), c-Myc and osteopontin. In non-small-cell lung cancer (NSCLC), levels of eIF4E are significantly increased compared with normal lung tissue. Here, we used an antisense oligonucleotide (ASO) to inhibit the expression of eIF4E in NSCLC cell lines. eIF4E levels were significantly reduced in a dose-dependent manner in NSCLC cells treated with eIF4E-specific ASO (4EASO) compared with control ASO. Treatment of NSCLC cells with the 4EASO resulted in decreased cap-dependent complex formation, decreased cell proliferation and increased sensitivity to gemcitabine. At the molecular level, repression of eIF4E with ASO resulted in decreased expression of the oncogenic proteins VEGF, c-Myc and osteopontin, whereas expression of ß-actin was unaffected. Based on these findings, we conclude that eIF4E-silencing therapy alone or in conjunction with chemotherapy represents a promising approach deserving of further investigation in future NSCLC clinical trials.

Gene therapy of established mesothelioma xenografts with recombinant p16INK4a adenovirus.

The absence of expression of the p16INK4a gene product is observed in virtually all mesothelioma tumors and cell lines, whereas wild-type pRB expression is maintained. We have examined the potential therapeutic role of re-expressing the p16INK4a gene product in mice with established human mesothelioma xenografts. Experiments using Adp16 treatments in mesothelioma xenografts demonstrated prolonged survival and potential cure following treatment with p16INK4a-based gene therapy. These results demonstrate that p16INK4a gene transfer may play a therapeutic role in the treatment of mesothelioma.