Circulating hormones and breast cancer risk in premenopausal women: a randomized trial of low-dose tamoxifen and fenretinide.

Tamoxifen and fenretinide have been extensively studied and exhibit breast cancer-preventing activity. We aimed to assess their effect on sex hormones, sex hormone binding globulin (SHBG) and retinol, and their association with mammographic density (MD) and breast cancer events. In a double-blind, placebo-controlled trial, premenopausal women at risk for breast cancer were randomized to tamoxifen 5 mg/day, fenretinide, both agents, or placebo for 2 years. We measured MD and circulating concentrations of follicle-stimulating hormone, luteinizing hormone (LH), estradiol, progesterone, testosterone, androstenedione, dehydro-epiandrosteronesulfate, prolactin, SHBG, and retinol at baseline and on yearly intervals. The associations with breast cancer events were evaluated through competing risk and Cox regression survival models. Low-dose tamoxifen markedly and enduringly increased SHBG, whereas the increases in testosterone, estradiol, and prolactin and reduction in LH weakened after 1 year. Fenretinide increased testosterone and androstenedione and decreased retinol. MD correlated directly with SHBG and inversely with retinol. After a median follow-up of 12 years, the 10-year cumulative incidence of breast cancer events was 37 % in women with SHBG ≤ 59.3 nmol/L, 22 % in women with SHBG between 59.3 and 101 nmol/L, and 19 % in women with SHBG > 101 nmol/L (P = 0.018). The difference among SHBG tertiles remained statistically significant at multivariable analysis: HR = 2.26 (95 % CI 1.04, 4.89) for the lowest versus the highest tertile. We conclude that low-dose tamoxifen or fenretinide exhibits favorable hormonal profiles as single agents, further supporting their administration for prevention of breast cancer in premenopause. Notably, SHBG levels were inversely associated with breast neoplastic events.

Human CD34+ cells engineered to express membrane-bound tumor necrosis factor-related apoptosis-inducing ligand target both tumor cells and tumor vasculature.

Adenovirus-transduced CD34+ cells expressing membrane-bound tumor necrosis factor-related apoptosis-inducing ligand (CD34-TRAIL+ cells) exert potent antitumor activity. To further investigate the mechanism(s) of action of CD34-TRAIL+ cells, we analyzed their homing properties as well as antitumor and antivascular effects using a subcutaneous myeloma model in immunodeficient mice. After intravenous injection, transduced cells homed in the tumor peaking at 48 hours when 188 plus or minus 25 CD45+ cells per 10(5) tumor cells were detected. Inhibition experiments showed that tumor homing of CD34-TRAIL+ cells was largely mediated by vascular cell adhesion molecule-1 and stromal cell-derived factor-1. Both CD34-TRAIL+ cells and soluble (s)TRAIL significantly reduced tumor volume by 40% and 29%, respectively. Computer-aided analysis of TdT-mediated dUTP nick end-labeling-stained tumor sections demonstrated significantly greater effectiveness for CD34-TRAIL+ cells in increasing tumor cell apoptosis and necrosis over sTRAIL. Proteome array analysis indicated that CD34-TRAIL+ cells and sTRAIL activate similar apoptotic machinery. In vivo staining of tumor vasculature with sulfosuccinimidyl-6-(biotinamido) hexanoate-biotin revealed that CD34-TRAIL+ cells but not sTRAIL significantly damaged tumor vasculature, as shown by TdT-mediated dUTP nick end-labeling+ endothelial cells, appearance of hemorrhagic areas, and marked reduction of endothelial area. These results demonstrate that tumor homing of CD34-TRAIL+ cells induces early vascular disruption, resulting in hemorrhagic necrosis and tumor destruction.

PLAB induction in fenretinide-induced apoptosis of ovarian cancer cells occurs via a ROS-dependent mechanism involving ER stress and JNK activation.

Fenretinide [N-(4-hydroxyphenyl)-retinamide (4HPR)] is a synthetic retinoid with antitumor activity that induces apoptosis in various types of cancer cell. We showed previously that 4HPR upregulates the proapoptotic gene placental bone morphogenetic protein (PLAB), which is a mediator of 4HPR-induced apoptosis in ovarian cancer cells. Here, we investigated the signaling cascade involving PLAB that mediates the apoptotic effect. In 4HPR-sensitive ovarian cancer cells, 4HPR-induced reactive oxygen species (ROS) are involved in PLAB upregulation and apoptosis, both events abrogated by the antioxidants vitamin C and butylated hydroxyanisole. We analyzed the expression and activation of endoplasmic reticulum (ER) stress-associated molecules and show that 4HPR-induced ER stress is a consequence of ROS generation. Salubrinal, an ER stress inhibitor, abrogated 4HPR-induced PLAB upregulation and protected the cells from apoptosis. Downstream of ROS generation and ER stress, 4HPR activated c-Jun N-terminal kinase (JNK), which was inhibited by vitamin C and salubrinal. The JNK inhibitor SP600125 reduced 4HPR-induced PLAB upregulation, by decreasing PLAB mRNA half-life, and protected the cells from apoptosis. These data indicate that 4HPR-induced PLAB upregulation occurs downstream of a signaling cascade involving ROS generation, ER stress induction and JNK activation and that these steps are mediators of 4HPR-induced apoptosis.

Valproic acid enhances bosutinib cytotoxicity in colon cancer cells.

Unbalanced histone deacetylase (HDAC) hyperactivity is a common feature of tumor cells. Inhibition of HDAC activity is often associated with cancer cell growth impairment and death. Valproic acid (VPA) is a HDAC inhibitor used for the treatment of epilepsy. It has recently been recognized as a promising anticancer drug. We investigated the effects of VPA on growth and survival of colon cancer cells. VPA caused growth inhibition and programmed cell death that correlated with histone hyperacetylation. VPA modulated the expression of various factors involved in cell cycle control and apoptosis and induced caspase activation. Interestingly, VPA induced downregulation of c-Src and potentiated the cytotoxic effects of the c-Src inhibitor bosutinib, both in vitro and in vivo. The combination of sublethal doses of VPA and bosutinib led to massive apoptosis of colon cancer cells, irrespective of their genetic background. These results suggest that VPA may be employed as a positive modulator of bosutinib antitumor activity in colorectal cancer.

Plasma retinol and prognosis of postmenopausal breast cancer patients.

BACKGROUND: The role of retinol (vitamin A) in breast cancer prognosis has never been investigated in postmenopausal women. We prospectively assessed the long-term prognostic role of retinol plasma levels in a cohort of postmenopausal breast cancer patients. PATIENTS AND METHODS: We investigated 208 women self-reported as postmenopausal operated on for T(1-2)N(0)M(0) breast cancer who participated in a chemoprevention trial as controls and never received chemotherapy or hormone therapy. Plasma samples were collected 3 months (median) after surgery and assayed within 3 weeks for retinol. Minimum and median potential follow-up were 12 and 15 years, respectively. The main analyses were on all women and on a subgroup ages >or=55 years, assumed too old to be in perimenopause. The main end point was breast cancer death. Breast cancer survival was estimated by the Kaplan-Meier method. The hazard ratios of breast cancer death by retinol level were estimated by Cox models stratified for age, where relevant, and recruitment period, and adjusted for tumor size and histology. RESULTS: At 12 years, patients with low retinol (<2.08 micromol/L, median of distribution) had lower breast cancer survival than those with high retinol (log-rank P = 0.052); the difference was significant for women >or=55 years (log-rank P = 0.006). The adjusted hazard ratios for low versus high retinol were 2.11 (95% confidence interval, 1.08-4.14) for all women and 3.58 (95% confidence interval, 1.50-8.57) for those >or=55 years. CONCLUSIONS: Low plasma retinol strongly predicts poorer prognosis in postmenopausal breast cancer patients. Retinol levels should be determined as part of the prognostic workup.

IFN-gamma enhances the antimyeloma activity of the fully human anti-human leukocyte antigen-DR monoclonal antibody 1D09C3.

To investigate the therapeutic activity of the fully human anti-HLA-DR antibody 1D09C3 in multiple myeloma (MM), we reevaluated HLA-DR expression on CD138(+) cells, analyzed the capacity of IFN-gamma to up-regulate HLA-DR expression on MM cell lines, and tested the in vitro and in vivo activity of 1D09C3 alone or in combination with IFN-gamma. CD138(+)HLA-DR(+) cells were detected in 31 of 60 patients, with 15 of 60 patients having >/=20% CD138(+)HLA-DR(+) cells (median, 50%; range, 23-100). Because primary plasma cells cannot be efficiently cultured in vitro, we used a panel of MM cell lines with a dim/negative to bright HLA-DR expression to evaluate 1D09C3-induced cell death. Annexin V/propidium iodide (PI) staining showed that 1D09C3-induced cell death correlated with constitutive HLA-DR expression. Induction of HLA-DR by IFN-gamma restored the sensitivity of HLA-DR dim cell lines to 1D09C3. In vivo, the combined IFN-gamma/1D09C3 treatment significantly increased the median survival of nonobese diabetic/severe combined immunodeficient mice xenografted with KMS-11 cell line, compared with controls (147 versus 48 days, P

Metallothionein 1G acts as an oncosupressor in papillary thyroid carcinoma.

The molecular pathogenesis of tumors arising from the thyroid follicular epithelial cells, including papillary (PTC) and follicular thyroid carcinoma (FTC), is only partially understood, and the role of tumor suppressor genes has not yet been assessed. The metallothionein (MT) gene family encodes a class of metal-binding proteins involved in several cellular processes, and their expression is often deregulated in human tumors. Recently, downregulation of MT gene expression in PTC has been reported, suggesting a possible oncosuppressor role of this gene family in the pathogenesis of thyroid tumors. To further explore this possibility, we performed expression and functional studies. Analysis of microarray data of thyroid tumors of different histologic types showed that several MT genes were downregulated with respect to normal tissue. The microarray data were corroborated by quantitative PCR experiments, showing downregulation of MTs in PTC and FTC, but to a greater extent in papillary carcinoma. The expression of MTs was also investigated at the protein level by immunohistochemistry; the results were consistent with the microarray data, showing general downregulation in tumor samples, which was more evident in PTC. The functional consequence of MT downregulation was addressed employing an experimental model made of the PTC-derived K1 cell line in which MT1G expression is repressed by promoter methylation. Restoration of MT1G expression by cDNA transfection affected growth rate and in vivo tumorigenicity of K1 cells, indicating an oncosuppressor role for MT1G in thyroid papillary tumorigenesis.

Characterization of compound 584, an Abl kinase inhibitor with lasting effects.

BACKGROUND: Resistance to imatinib is an important clinical issue in the treatment of Philadelphia chromosome-positive leukemias which is being tackled by the development of new, more potent drugs, such as the dual Src/Abl tyrosine kinase inhibitors dasatinib and bosutinib and the imatinib analog nilotinib. In the current study we describe the design, synthesis and biological properties of an imatinib analog with a chlorine-substituted benzamide, namely compound 584 (cmp-584). DESIGN AND METHODS: To increase the potency, we rationally designed cmp-584, a compound with enhanced shape complementarity with the kinase domain of Abl. cmp-584 was synthesized and characterized in vitro against a panel of 67 serine/threonine and tyrosine kinases using radioactive and enzyme-linked immunosorbent kinase assays. We studied inhibitory cellular activity using Bcr/Abl-positive human cell lines, murine transfectants in proliferation experiments, and a murine xenotrans-planted model. Kinase assays on isolated Bcr/Abl protein were also performed. Finally, we used a wash-out approach on whole cells to study the binding kinetics of the inhibitor. RESULTS: cmp-584 showed potent anti-Abl activity both on recombinant protein (IC(50): 8 nM) and in cell-based assays (IC(50): 0.1-10 nM). The drug maintained inhibitory activity against platelet-derived growth factor receptors and c-KIT and was also active against Lyn (IC(50): 301 nM). No other kinase of the panel was inhibited at nanomolar doses. cmp-584 was 20- to 300-fold more active than imatinib in cells. This superior activity was evident in intact cells, in which full-length Bcr-Abl is present. In vivo experiments confirmed the activity of cmp-584. Wash-out experiments showed that short exposure to the drug impaired cell proliferation and Bcr-Abl phosphorylation for a substantially longer period of time than imatinib. CONCLUSIONS: The present results suggest a slower off-rate (dissociation rate) of cmp-584 compared to imatinib as an explanation for the increased cellular activity of the former.

Pharmacokinetics of oral fenretinide in neuroblastoma patients: indications for optimal dose and dosing schedule also with respect to the active metabolite 4-oxo-fenretinide.

PURPOSE: Pharmacokinetic data on fenretinide (4-HPR) are scant, thus limiting the rational use of the drug. We investigated the pharmacokinetics of 4-HPR and its active metabolite 4-oxo-fenretinide (4-oxo-4-HPR). EXPERIMENTAL DESIGN: Pharmacokinetics were assessed in 18 children (3 for each dose) with neuroblastoma who received oral 4-HPR once daily for 28 days at the doses of 100, 300, 400, 600, 1,700 and 4,000 mg/m(2)/day. 4-HPR and 4-oxo-4-HPR were determined by HPLC in plasma collected up to 48 h after the first and 28th administration. RESULTS: After single administration, 4-HPR mean C (max) ranged from 0.9 to 6.6 microM and these concentrations roughly doubled at steady state (range 1.6-14.5 microM). 4-HPR mean t (1/2) was 22 h. 4-HPR pharmacokinetics were linear in the dose range 100-1,700 mg/m(2); less than dose-proportional increase in exposure was found at 4,000 mg/m(2). At steady state, pharmacologically relevant plasma concentrations (range 0.7-10 microM and 0.4-5 microM for 4-HPR and 4-oxo-4-HPR, respectively) were maintained during the 24 h dosing interval in the dose range 300-4,000 mg/m(2). CONCLUSIONS: 4-HPR pharmacokinetics supports once-daily dosing. Steady state concentrations of 4-HPR and 4-oxo-4-HPR in children with neuroblastoma are in line with those found to have in vitro growth inhibitory effects in neuroblastoma cells.

4-oxo-fenretinide, a recently identified fenretinide metabolite, induces marked G2-M cell cycle arrest and apoptosis in fenretinide-sensitive and fenretinide-resistant cell lines.

4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) is a recently identified metabolite of fenretinide (4-HPR). We explored the effectiveness of 4-oxo-4-HPR in inducing cell growth inhibition in ovarian, breast, and neuroblastoma tumor cell lines; moreover, we investigated the molecular events mediating this effect in two ovarian carcinoma cell lines, one sensitive (A2780) and one resistant (A2780/HPR) to 4-HPR. 4-oxo-4-HPR was two to four times more effective than 4-HPR in most cell lines, was effective in both 4-HPR-sensitive and 4-HPR-resistant cells, and, in combination with 4-HPR, caused a synergistic effect. The tumor growth-inhibitory effects of 4-oxo-4-HPR seem to be independent of nuclear retinoid receptors (RAR), as indicated by the failure of RAR antagonists to inhibit its effects and by its poor ability to bind and transactivate RARs. Unlike 4-HPR, which only slightly affected the G(1) phase of the cell cycle, 4-oxo-4-HPR caused a marked accumulation of cells in G(2)-M. This effect was associated with a reduction in the expression of regulatory proteins of G(2)-M (cyclin-dependent kinase 1 and cdc25c) and S (cyclin A) phases, and with an increase in the expression of apoptosis-related proteins, such as p53 and p21. Apoptosis was induced by 4-oxo-4-HPR in both 4-HPR-sensitive and 4-HPR-resistant cells and involved activation of caspase-3 and caspase-9 but not caspase-8. We also showed that 4-oxo-4-HPR, similarly to 4-HPR, increased reactive oxygen species generation and ceramide levels by de novo synthesis. In conclusion, 4-oxo-4-HPR is an effective 4-HPR metabolite that might act as therapeutic agent per se and, when combined with 4-HPR, might improve 4-HPR activity or overcome 4-HPR resistance.

The anti-human leukocyte antigen-DR monoclonal antibody 1D09C3 activates the mitochondrial cell death pathway and exerts a potent antitumor activity in lymphoma-bearing nonobese diabetic/severe combined immunodeficient mice.

The fully human anti-HLA-DR antibody 1D09C3 has been shown to delay lymphoma cell growth in severe combined immunodeficient (SCID) mice. The present study was aimed at (a) investigating the mechanism(s) of 1D09C3-induced cell death and (b) further exploring the therapeutic efficacy of 1D09C3 in nonobese diabetic (NOD)/SCID mice. The chronic lymphocytic leukemia cell line JVM-2 and the mantle cell lymphoma cell line GRANTA-519 were used. Generation of reactive oxygen species (ROS) and mitochondrial membrane depolarization were measured by flow cytometry following cell incubation with dihydroethidium and TMRE, respectively. Western blot analysis was used to detect c-Jun-NH(2)-kinase (JNK) phosphorylation and apoptosis-inducing factor (AIF). NOD/SCID mice were used to investigate the activity of 1D09C3 in early- or advanced-stage tumor xenografts. In vitro, 1D09C3-induced cell death involves a cascade of events, including ROS increase, JNK activation, mitochondrial membrane depolarization, and AIF release from mitochondria. Inhibition of JNK activity significantly reduced 1D09C3-induced apoptosis, indicating that 1D09C3 activity involves activation of the kinase. In vivo, 1D09C3 induces long-term disease-free survival in a significant proportion of tumor-bearing mice treated at an early stage of disease. Treatment of mice bearing advanced-stage lymphoma results in a highly significant prolongation of survival. These data show that 1D09C3 (a) exerts a potent antitumor effect by activating ROS-dependent, JNK-driven cell death, (b) cures the great majority of mice treated at an early-stage of disease, and (c) significantly prolongs survival of mice with advanced-stage disease.

In vitro and in vivo activity of SKI-606, a novel Src-Abl inhibitor, against imatinib-resistant Bcr-Abl+ neoplastic cells.

Resistance to imatinib represents an important scientific and clinical issue in chronic myelogenous leukemia. In the present study, the effects of the novel inhibitor SKI-606 on various models of resistance to imatinib were studied. SKI-606 proved to be an active inhibitor of Bcr-Abl in several chronic myelogenous leukemia cell lines and transfectants, with IC(50) values in the low nanomolar range, 1 to 2 logs lower than those obtained with imatinib. Cells expressing activated forms of KIT or platelet-derived growth factor receptor (PDGFR), two additional targets of imatinib, were unaffected by SKI-606, whereas activity was found against PIM2. SKI-606 retained activity in cells where resistance to imatinib was caused by BCR-ABL gene amplification and in three of four Bcr-Abl point mutants tested. In vivo experiments confirmed SKI-606 activity in models where resistance was not caused by mutations as well as in cells carrying the Y253F, E255K, and D276G mutations. Modeling considerations attribute the superior activity of SKI-606 to its ability to bind a conformation of Bcr-Abl different from imatinib.

CD52 antigen expressed by malignant plasma cells can be targeted by alemtuzumab in vivo in NOD/SCID mice.

OBJECTIVE: To explore new treatments specifically targeting malignant plasma cells (PCs), we examined CD52 antigen expression on primary PCs as well as multiple myeloma (MM) cell lines, and investigated in vivo the antimyeloma activity of alemtuzumab. MATERIALS AND METHODS: PCs were enriched from the marrow of MM patients (n = 39) according to CD138 expression and then analyzed by 3-color flow cytometry and quantitative PCR. The in vivo activity of alemtuzumab was evaluated in a xenotransplant model of MM in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. RESULTS: CD52 expression revealed a substantial heterogeneity in terms of both percentage of positive cells and fluorescence intensity, with 25/39 (64%) MM patients showing >or=30% CD138(+) PCs expressing the CD52 antigen (mean = 79%; range, 33-100%). Similarly to primary cells, cell lines showed heterogeneous CD52 expression. Expression of CD52 mRNA by quantitative PCR analysis strongly correlated with CD52 antigen detection by flow cytometry. In vivo, alemtuzumab treatment significantly increased the median survival of animals with an early- (64 vs 77 days, p

A phase I-II preoperative biomarker trial of fenretinide in ascitic ovarian cancer.

PURPOSE: To evaluate study feasibility, toxicity, drug concentrations, and activity of escalating doses of the synthetic retinoid fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] in ovarian cancer by measuring serum CA125 and cytomorphometric biomarkers in cancer cells collected from ascitic fluid before and after treatment. METHODS: Twenty-two naive patients with ascitic ovarian cancer were treated with escalating doses of 4-HPR at 0, 400, 600, and 800 mg/d for 1 to 4 weeks before surgery. Changes in the proportion of proliferating cells expressed by Ki67 and computer-assisted cytomorphometric variables (nuclear area, DNA index, and chromatin texture) were determined in ascitic cells. Drug levels were measured by high-performance liquid chromatography. RESULTS: Doses up to 800 mg/d were well tolerated, and no adverse reactions occurred. There was no effect of 4-HPR on changes in serum CA125, Ki67 expression, which were assessed in 75% of subjects, and cytomorphometric variables, which were assessed in 80% of subjects. Plasma retinol levels were significantly lower in affected women than healthy donors. 4-HPR plasma concentrations increased slightly with increasing doses and attained a 1.4 micromol/L concentration with 800 mg/d. Drug levels in malignant ascitic cells and tumor tissue were higher than in plasma but were 50 and 5 times lower, respectively, than in carcinoma cells treated in vitro with 1 micromol/L 4-HPR. CONCLUSIONS: Cell biomarkers can be measured in ascitic cells to assess drug activity. Under our experimental conditions, 4-HPR did not show activity in advanced ovarian cancer cells. However, clinical evidence supports further investigation of fenretinide for ovarian cancer prevention.

Inhibition of RET tyrosine kinase by SU5416.

Thyroid neoplasia is frequently associated with rearranged during transfection (RET) proto-oncogene mutations that cause hyperactivation of RET kinase activity. Selective inhibition of RET-mediated signaling should lead to an efficacious therapy. SU5416 is a potent inhibitor of vascular endothelial cell growth factor receptor, c-Kit, and FLT-3 receptor tyrosine kinases presently used in clinical trials. We found that SU5416 inhibits RET with similar potency, both in cell-free assays and in cells, thus causing proliferation arrest in oncogenic RET-transfected cells and in papillary thyroid carcinoma (PTC) cells expressing the RET/PTC1 oncogene, but not in RET-negative control cells. SU5416 inhibited RET-mediated signaling through the extracellular signal regulated kinase (ERK) and JNK pathways. In addition, we show that a naturally occurring MEN2 mutation at codon 804 confers resistance to SU5416, but not to the related compound SU4984. We provide a possible explanation to these results by using molecular docking. Finally, SU5416 was also assessed against an array of 52 tyrosine and serine/threonine kinases.

Defibrotide in combination with granulocyte colony-stimulating factor significantly enhances the mobilization of primitive and committed peripheral blood progenitor cells in mice.

Defibrotide is a polydeoxyribonucleotide, which significantly reduces the expression of adhesion molecules on endothelial cells. We investigated the activity of Defibrotide alone or in combination with recombinant human granulocyte colony-stimulating factor (rhG-CSF) to mobilize peripheral blood progenitor cells (PBPCs) in BALB/c mice. A 5-day treatment with Defibrotide alone (1-15 mg/mouse/day) had no effect on WBC counts, frequencies and absolute numbers of total circulating colony-forming cells (CFCs), i.e., granulocyte-macrophage colony-forming units, erythroid burst-forming units, and multilineage colony-forming units. As compared with mock-injected mice, administration of rhG-CSF alone (5 micro g/mouse/day) for 5 days significantly (P < or = 0.0001) increased WBC counts, CFC frequencies, and CFC absolute numbers by 2-, 13-, and 27-fold, respectively. As compared with control mice, the combined administration of Defibrotide (15 mg/mouse/day) and rhG-CSF significantly (P < or = 0.0001) increased WBC counts, frequencies and absolute numbers of CFCs by 4-, 38-, and 119-fold, respectively. As compared with rhG-CSF alone, administration of Defibrotide plus rhG-CSF resulted in a significant increase (P < or = 0.001) of the frequency of circulating long-term culture-initiating cells. In addition, transplantation of 2 x 10(5) rhG-CSF- or Defibrotide/rhG-CSF-mobilized mononuclear cells rescued 43% and 71% of recipient mice, respectively. Experiments of CFC homing performed in lethally irradiated or nonirradiated recipients showed that marrow homing of transplanted PBPCs was reduced by 3-fold in Defibrotide-treated animals as compared with mock-injected mice (P < or = 0.001), suggesting that the mobilizing effect of Defibrotide might be because of an effect on PBPC trafficking. In conclusion, our data demonstrate that Defibrotide synergizes with rhG-CSF and significantly increases the mobilization of a broad spectrum of PBPCs, including primitive and committed progenitor cells. These data might have relevant implications for autologous and allogeneic anticancer therapy in humans.

Role of TFG sequences outside the coiled-coil domain in TRK-T3 oncogenic activation.

The TRK-T3 oncoprotein, isolated from a human papillary thyroid tumor, arises from the fusion between the N-terminal domain of the TFG gene and the tyrosine kinase domain of the NTRK1 receptor. The 68 kDa TRK-T3 oncoprotein displays a constitutive tyrosine kinase activity resulting in its capability to transform NIH3T3 cells. The TFG portion of TRK-T3 contains a coiled-coil domain, which mediates protein oligomerization essential for the oncogene constitutive activation, and several consensus sites for protein interaction. In this study, we investigate the role of TFG sequences outside the coiled-coil domain on TRK-T3 activation, We constructed four mutants carrying different deletions of TFG sequences and expressed them in mammalian cells. By performing biochemical and biological assays we demonstrated that all the deleted regions are required for TRK-T3 activation, as they are involved in different mechanisms such as protein processing, formation of stable and/or functional complexes, and possible interaction with other proteins. By constructing site-specific mutants, we demonstrated a crucial role for a PB1 domain and a considerable contribution of an SH2-binding motif in TRK-T3 oncogenic activation. This work establishes an important role for TFG sequences outside the coiled-coil domain in the activation of the thyroid TRK-T3 oncogene.

Different ways to induce apoptosis by fenretinide and all-trans-retinoic acid in human B lymphoma cells.

All-trans-retinoic acid (ATRA) and its synthetic analog fenretinide (4HPR) are potent anticancer drugs. Only a few reports are available about the effects of retinoids on B lymphoma cells. In our study, non-Hodgkin lymphoma cells (HT58) were treated with ATRA and 4HPR. Both agents induced cell death time- and dose-dependently. Reactive oxygen species (ROS) production was elevated in 4HPR-treated cells, but not in ATRA-treated cells. The depolarization of the mitochondrial membrane occured earlier after ATRA than after 4HPR treament. Z-VAD-fmk, the general caspase inhibitor, decreased the DNA fragmentation in ATRA-treated cells, but simultaneously increased necrosis. However, z-VAD-fmk did not influence the DNA fragmentation in 4HPR-treated cells. Endonuclease G was released from the mitochondria during 4HPR treatment, which could be an inducer for caspase-independent DNA fragmentation. Our results suggest that natural (ATRA) and synthetic (4HPR) retinoids induce different apoptotic pathways in B lymphoma cells, which is particularly relevant for their potential use in leukemia treatment.

Identification of the fenretinide metabolite 4-oxo-fenretinide present in human plasma and formed in human ovarian carcinoma cells through induction of cytochrome P450 26A1.

PURPOSE: The synthetic retinoid fenretinide (4-HPR) exhibits preventive and therapeutic activity against ovarian tumors. An unidentified polar metabolite was previously found in 4-HPR-treated subjects and in A2780 human ovarian carcinoma cells continuously treated with 4-HPR (A2780/HPR). The metabolite and the enzyme involved in its formation in tumor cells are herein identified. EXPERIMENTAL DESIGN: The metabolite was identified by mass spectrometry in A2780/HPR cell extracts and in plasma from 11 women participating in a phase III trial and treated with 200 mg/d 4-HPR for 5 years. The expression of proteins involved in retinoid metabolism and transport, cytochrome P450 26A1 (CYP26A1), cellular retinol-binding protein I (CRBP-I), and cellular retinoic acid-binding protein I and II (CRABP-I, CRABP-II) were evaluated in tumor cells by reverse transcription-PCR and Western blot analyses. Overexpression of CYP26A1 and retinoic acid receptors (RARs) in A2780 cells were obtained by cDNAs transfection. RESULTS: The polar metabolite was 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) i.e., an oxidized form of 4-HPR with modification in position 4 of the cyclohexene ring. 4-oxo-4-HPR plasma levels were slightly lower (0.52 +/- 0.17 micromol/L) than those of the parent drug (0.84 +/- 0.53 micromol/L) and of the already identified metabolite N-(4-methoxyphenyl)retinamide (1.13 +/- 0.85 micromol/L). In A2780/HPR cells continuously treated with 4-HPR and producing 4-oxo-4-HPR, CYP26A1 and CRBP-I were markedly up-regulated compared with A2780 untreated cells. In A2780 cells, not producing 4-oxo-4-HPR, overexpression of CYP26A1 caused formation of 4-oxo-4-HPR, which was associated with no change in 4-HPR sensitivity. Moreover, the addition of 4-oxo-4-HPR to A2780 cells inhibited cell proliferation. Elevated levels of CYP26A1 protein and metabolism of 4-HPR to 4-oxo-4-HPR were found in A2780 cells transfected with RARbeta and to a lesser extent in those transfected with RARgamma. CONCLUSIONS: A new metabolite of 4-HPR, 4-oxo-4-HPR, present in human plasma and in tumor cells, has been identified. The formation of this biologically active metabolite in tumor cells was due to CYP26A1 induction and was influenced by RAR expression. Moreover evidence was provided that 4-HPR up-modulates the expression of CRBP-I transcript, which is lost during ovarian carcinogenesis.

Relationships between plasma insulin-like growth factor-I and insulin-like growth factor binding protein-3 and second breast cancer risk in a prevention trial of fenretinide.

PURPOSE: High circulating insulin-like growth factor (IGF) -I and/or low IGF-binding protein (IGFBP) -3 levels are associated with increased breast cancer risk in unaffected premenopausal women. We determined whether IGF-I and IGFBP-3 predict second breast cancer risk, and whether their changes during fenretinide explain observed reductions in second breast cancer in women