TIMP3 regulates migration, invasion and in vivo tumorigenicity of thyroid tumor cells.

Papillary thyroid carcinoma (PTC) arises from the thyroid follicular epithelium and represents the most frequent thyroid malignancy. PTC is associated with gene rearrangements generating RET/PTC and TRK oncogenes, and to the BRAFV600E activating point mutation. A role of tumor-suppressor genes in the pathogenesis of PTC has not been assessed yet. The tissue inhibitor of metalloproteinase-3 (TIMP3) gene, encoding a metalloproteinases inhibitor and capable of inhibiting growth, angiogenesis, invasion and metastasis of several cancers, was found to be silenced by promoter methylation in a consistent fraction of PTCs, in association with tumor aggressiveness and BRAFV600E mutation, thus suggesting an oncosuppressor role. To explore this possibility, in this study we performed gene expression and functional studies. Analysis of gene expression data produced in our laboratory as well as meta-analysis of publicly available data sets confirmed the downregulation of TIMP3 gene expression in PTC with respect to normal thyroid. The functional consequences of TIMP3 downregulation were investigated in the PTC-derived NIM1 cell line, in which the expression of TIMP3 is silenced. Restoration of TIMP3 expression by exposure to soluble TIMP3 protein or by complementary DNA transfection had no effect on the growth rate of NIM1 cells. Instead, it affected the adhesive, migratory and invasive capabilities of NIM1 cells by modulating several proteins involved in these processes. A striking effect was observed in vivo, as TIMP3 reduced the tumorigenicity of NIM1 cells by repressing angiogenesis and macrophage infiltration. Our data indicate that the loss of TIMP3 expression exerts a functional role in the pathogenesis of PTC.

IGFBP7: an oncosuppressor gene in thyroid carcinogenesis.

Insulin-like growth factor-binding protein 7 (IGFBP7) is a secreted protein involved in several cellular processes, including proliferation, senescence and apoptosis. Loss of IGFBP7 expression is a critical step in the development of human tumors, including melanoma and colon cancer. By microarray gene expression studies, we have detected downregulation of IGFBP7 gene expression in follicular and papillary thyroid tumors in comparison with normal thyroid tissue. Evaluation of publicly available PTC microarray gene expression data sets confirmed, in a consistent fraction of tumors, the downregulation of IGFBP7 transcript levels. The functional consequence of IGFBP7 downregulation was addressed in the PTC-derived NIM1 cell line in which IGFBP7 expression is repressed by promoter hypermethylation. Exposure to soluble IGFBP7 protein or restoration of IGFBP7 expression by complementary DNA transfection reduced growth rate, migration, anchorage-independent growth and tumorigenicity of NIM1 cells. We show that the effects of IGFBP7 are related to apoptosis. Our data suggest that loss of IGFBP7 expression has a functional role in thyroid carcinogenesis, and it may represent a possible basis for therapeutic strategies.

Analysis of gene expression identifies PLAB as a mediator of the apoptotic activity of fenretinide in human ovarian cancer cells.

Fenretinide (4-HPR) is a synthetic retinoid with antitumor activity, which induces apoptosis in cancer cell lines of different histotypes. To identify genes contributing to its apoptotic activity in ovarian cancer cells, we monitored, by cDNA arrays, gene expression changes after 4-HPR exposure in A2780, a human ovarian carcinoma cell line sensitive to the retinoid. Among the differentially expressed transcripts, PLAcental Bone morphogenetic protein (PLAB), a proapoptotic gene, was the most highly induced. In a panel of ovarian carcinoma cell lines with different 4-HPR sensitivities, PLAB upregulation was associated with cellular response to 4-HPR, its overexpression increased basal apoptosis and its silencing by small interfering RNA decreased the ability of 4-HPR to induce apoptosis. PLAB induction by 4-HPR was p53- and EGR-1 independent and was regulated, at least in part, by increased stability of PLAB mRNA. PLAB up-modulation by 4-HPR also occurred in vivo: in ascitic cells collected from patients with ovarian cancer before and after 4-HPR treatment, PLAB was upmodulated in 2/4 patients. Our results in certain ovarian cancer cell lines indicate a role for PLAB as a mediator of 4-HPR-induced apoptosis. The correlation of increased PLAB in vivo with antitumor activity remains to be established.

Fifteen-year results of a randomized phase III trial of fenretinide to prevent second breast cancer.

PURPOSE: The synthetic retinoid fenretinide administered for 5 years for prevention of second breast cancer showed no difference after a median of 8 years, but a possible reduction in premenopausal women. We conducted a long-term analysis in a subgroup of women who were regularly followed up in a single center. PATIENTS AND METHODS: We analyzed data after a median follow-up of 14.6 years (IQ range, 12.3-16.3 years) from 1739 women aged 30-70 (872 in the fenretinide arm and 867 in the observation arm), representing 60% of the initial cohort of 2867 women. The main efficacy endpoint was second primary breast cancer (contralateral or ipsilateral). RESULTS: The number of second breast cancers was 168 in the fenretinide arm and 190 in the control arm (hazard ratio = 0.83, 95% CI, 0.67-1.03). There were 83 events in the fenretinide arm and 126 in the observation arm in premenopausal women (HR = 0.62, 95% CI, 0.46-0.83), and 85 and 64 events in postmenopausal women (HR = 1.23, 95% CI, 0.63-2.40). The younger were the women, the greater was the risk reduction associated with fenretinide, which attained 50% in women aged 40 years or younger and disappeared after age 55 (P-age*treatment interaction = 0.023). There was no difference in cancers in other organs, distant metastases or survival. CONCLUSIONS: Fenretinide induces a significant risk reduction of second breast cancer in premenopausal women, which is remarkable at younger ages, and persists several years after treatment cessation. Since adverse events are limited, a trial in young women at high-risk is warranted.

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

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

Effect of imatinib on haematopoietic recovery following idarubicin exposure.

SCF is a potent pro-proliferative cytokine crucial for haematopoiesis, which binds to c-kit and activates its tyrosine kinase activity. Inactivating mutations of either SCF or c-kit have been described in mice and lead to increased sensitivity to treatment with ionising radiation. Imatinib is a tyrosine kinase inhibitor with high affinity for c-Abl, PDGFR and c-kit. In this study we investigated the effect of concomitant administration of imatinib and idarubicin, an anthracycline with haematosuppressive activity, in nu/nu mice and murine bone marrow cells. Double-treated animals showed significantly increased mortality compared to mice that received imatinib or idarubicin alone only when idarubicin and imatinib were given simultaneously. The combined treatment induced a more severe neutropenia with a slower recovery when compared to mice treated with idarubicin alone. The myeloid metaplasia usually observed in the spleen after idarubicin treatment was absent in mice co-treated with imatinib. Bone marrow from double-treated animals also showed decreased numbers of megakaryocytes and myeloid precursor cells. In vitro culture of murine bone marrow cells in the presence of imatinib inhibited SCF-induced proliferation and recovery from treatment with idarubicin. Our results indicate that the simultaneous administration of imatinib enhances idarubicin-induced haematopoietic toxicity in vivo and in vitro.

Long-term effects of fenretinide, a retinoic acid derivative, on the insulin-like growth factor system in women with early breast cancer.

High insulin-like growth factor-I (IGF-I) levels are associated with an increased risk of breast cancer in premenopausal women. Because the synthetic retinoid fenretinide showed a beneficial effect on second breast cancers in premenopausal women in a Phase III trial, we studied its long-term effects on IGF-I levels. We measured, at yearly intervals for up to 5 years, the circulating levels of IGF-I, IGF binding protein (BP)-3, and their molar ratio in 60 subjects < or = 50 years of age and 60 subjects > 50 years of age allocated either to fenretinide or no treatment. In women < or = 50 years of age, measurements of IGF-II, IGFBP-1, and IGFBP-2 were also performed. The associations between biomarkers and drug or metabolite plasma concentrations were also investigated. All biomarkers were relatively stable over 5 years in the control group. Compared with controls and after adjustment for baseline, treatment with fenretinide for 1 year induced the following changes: IGF-I, -13% [95% confidence interval (CI), -25 to 1%] in women < or = 50 years of age and -3% (95% CI, -16 to 13%) in women > 50 years of age; IGFBP-3, -4% (95% CI, -12 to 6%) in both age groups; IGF-I:IGFBP-3 molar ratio, -11% (95% CI, -22 to 1%) in women < or = 50 years of age and 1% (95% CI, -11 to 16%) in women > 50 years of age. These effects were apparently maintained for up to 5 years, although fewer samples were available as time progressed. No change in other IGF components was observed. Drug and metabolite concentrations were negatively correlated with IGF-I and IGF-I:IGFBP-3 molar ratio in women < or = 50 years of age. Fenretinide induces a moderate decline of IGF-I levels in women < or = 50 years of age. The association between IGF-I change and the reduction of second breast cancers in premenopausal women warrants further study.

Chemoprevention of breast cancer with fenretinide.

Chemoprevention of cancer represents a challenge for oncology during this new millennium. Substantial advances have been accomplished in the last decade, especially for primary and secondary prevention of breast cancer. In addition to tamoxifen, raloxifene and other selective estrogen receptor modulators, retinoids are among the most promising agents, given their ability to inhibit mammary carcinogenesis in preclinical models. Fenretinide, the synthetic amide of retinoic acid, inhibits cell growth mostly through the induction of apoptosis with mechanisms which may partly involve the retinoid receptors. Because it has a favourable toxicological profile, fenretinide has been extensively investigated in clinical trials. A large randomised phase III trial for secondary breast cancer prevention has been recently carried out in Italy. Results showed a reduction of second breast malignancies in premenopausal women. In addition, a significant decrease of circulating insulin-like growth factor (IGF)-1, a known risk factor for premenopausal breast cancer, was observed after 1 year of fenretinide administration in premenopausal women with breast cancer. Ongoing studies on the validation of the circulating IGF-1 as a surrogate endpoint biomarker of fenretinide activity and on the effectiveness of the combination with low dose tamoxifen may provide further insight into the future clinical application of fenretinide.

Decrease in drug accumulation and in tumour aggressiveness marker expression in a fenretinide-induced resistant ovarian tumour cell line.

We investigated whether the efficacy of fenretinide (HPR) against ovarian tumours may be limited by induction of resistance. The human ovarian carcinoma cell line A2780, which is sensitive to a pharmacologically achievable HPR concentration (IC(50)= 1 microM), became 10-fold more resistant after exposure to increasing HPR concentrations. The cells (A2780/HPR) did not show cross-resistance to the synthetic retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) and were not sensitive, similarly to the parent line, to all-trans-retinoic acid, 13-cis-retinoic acid or N-(4-methoxyphenyl)retinamide. A2780/HPR cells showed, compared to parental cells, a 3-fold reduction in colony-forming ability in agar. The development of HPR resistance was associated with a marked increase in retinoic acid receptor beta (RARbeta) mRNA and protein levels, which decreased, together with drug resistance, after drug removal. The expression of cell surface molecules associated with tumour progression including HER-2, laminin receptor and beta1 integrin was markedly reduced. The increase in the levels of reactive oxygen species is not involved in HPR-resistance because it was similar in parental and resistant cells. Conversely differences in pharmacokinetics may account for resistance because, in A2780/HPR cells, intracellular peak drug levels were 2 times lower than in A2780 cells and an as yet unidentified polar metabolite was present. These data suggest that acquired resistance to HPR is associated with changes in marker expression, suggestive of a more differentiated status and may be explained, at least in part, by reduced drug accumulation and increased metabolism.

Growth-inhibitory effect of STI571 on cells transformed by the COL1A1/PDGFB rearrangement.

Dermatofibrosarcoma protuberans (DP) is a skin tumor of intermediate malignancy characterized by high recurrence rates, for which surgical excision is the main therapy. All DP cases carry a specific t(17;22) translocation, resulting in a COL1A1/PDGFB rearrangement. The subsequently deregulated production of PDGFB generates autocrine stimulation of PDGFrbeta, leading to malignant transformation. Using NIH-3T3 cells transformed by the COL1A1/PDGFB rearrangement (5A cell line), we explored the possibility of blocking the PDGFB autocrine loop, both in vitro and in vivo, using STI571, an inhibitor of the PDGF receptor and of ABL kinase activity. The presence of small amounts of serum in the culture medium was required for the in vitro growth and morphological transformation of 5A cells. In the presence of STI571, the growth rate was reduced and the associated transformed phenotype changed to a flattened one. This effect could be reversed on removal of the inhibitor. The growth rate of tumors induced by 5A cells in nude mice was reduced by STI571 administration. Interestingly, this effect was also evident on pre-existing tumors, but no tumor eradication was observed. This is consistent with the reversible effects of the inhibitor observed in vitro but differs from the eradication effect of STI571 on BCR-ABL-induced tumors. Our data indicate that STI571 might be a candidate compound for the pharmacological treatment of DP and demonstrate that the same compound may act in different ways (cytotoxic vs. cytostatic), according to the specificity of the inhibited tyrosine kinase, namely, ABL or PDGFrbeta.

Therapeutic effects of the combination of fenretinide and all-trans-retinoic acid and of the two retinoids with cisplatin in a human ovarian carcinoma xenograft and in a cisplatin-resistant sub-line.

We previously showed that fenretinide (4-HPR), a synthetic derivative of all-traits retinoic acid (RA), is effective in mice bearing the human ovarian carcinoma IGROV-1 and it significantly enhances the antitumour activity of cisplatin on the same tumour. The present study examined the therapeutic effects of the combination of 4-HPR and RA and of the two retinoids with cisplatin as intracavitary treatments of mice bearing IGROV-1 and IGROV-1/cisplatin tumours, the latter derived from a sub-line with an in vivo reduced sensitivity to cisplatin. 4-HPR, as a single agent, was effective against both tumours, whereas RA had no effect. In IGROV-1 tumour-bearing mice, the combination of RA and 4-HPR significantly improved the efficacy of 4-HPR, resulting in an antitumour activity similar to that obtained with cisplatin alone. N-(4-methoxyphenylretinamide), the main metabolite of 4-HPR, had no antitumour effect and it did not increase 4-HPR activity in IGROV-1 tumour-bearing mice. In the same tumour model, 4-HPR and RA separately increased cisplatin activity, even though for RA the increase was not statistically significant. In contrast, the association of the two retinoids together with cisplatin did not produce any benefit and resulted in increased toxicity. In IGROV-1/cisplatin tumour-bearing mice, the association of 4-HPR (but not of RA) to cisplatin significantly increased cisplatin activity, resulting in the reversal of cisplatin resistance. These findings demonstrate that 4-HPR may be effective and enhance cisplatin sensitivity in cisplatin-sensitive and -resistant ovarian tumours and that the association of RA and 4-HPR may result in increased 4-HPR antitumour activity.

Role of alpha1 acid glycoprotein in the in vivo resistance of human BCR-ABL(+) leukemic cells to the abl inhibitor STI571.

BACKGROUND: Chronic myeloid leukemia is caused by a chromosomal translocation that results in an oncogenic fusion protein, Bcr-Abl. Bcr-Abl is a tyrosine kinase whose activity is inhibited by the antineoplastic drug STI571. This drug can cure mice given an injection of human leukemic cells, but treatment ultimately fails in animals that have large tumors when treatment is initiated. We created a mouse model to explore the mechanism of resistance in vivo. METHODS Nude mice were injected with KU812 Bcr-Abl(+) human leukemic cells. After 1 day (no evident tumors), 8 days, or 15 days (tumors >1 g), mice were treated with STI571 (160 mg/kg every 8 hours). Cells recovered from relapsing animals were used for in vitro experiments. Statistical tests were two-sided. RESULTS: Tumors regressed initially in all STI571-treated mice, but all mice treated 15 days after injection of tumor cells eventually relapsed. Relapsed animals did not respond to further STI571 treatment, and their Bcr-Abl kinase activity in vivo was not inhibited by STI571, despite high plasma concentrations of the drug. However, tumor cells from resistant animals were sensitive to STI571 in vitro, suggesting that a molecule in the plasma of relapsed animals may inactivate the drug. The plasma protein alpha1 acid glycoprotein (AGP) bound STI571 at physiologic concentrations in vitro and blocked the ability of STI571 to inhibit Bcr-Abl kinase activity in a dose-dependent manner. Plasma AGP concentrations were strongly associated with tumor load. Erythromycin competed with STI571 for AGP binding. When animals bearing large tumors were treated with STI571 alone or with a combination of STI571 and erythromycin, greater tumor reductions and better long-term tumor-free survival (10 of 12 versus one of 13 at day 180; P:<.001) were observed after the combination treatment. CONCLUSION: AGP in the plasma of relapsed animals binds to STI571, preventing this compound from inhibiting the Bcr/Abl tyrosine kinase. Molecules such as erythromycin that compete with STI571 for binding to AGP may enhance the therapeutic potential of this drug.

Quality control for HPLC assay and surrogate end point biomarkers from the fenretinide (4-HPR) breast cancer prevention trial.

The Fenretinide (4-HPR) Breast Cancer Study is a randomized multicenter clinical trial designed to evaluate the effectiveness of the synthetic retinoid 4-HPR, at a dose of 200 mg per os every day for 5 years, in reducing the incidence of contralateral breast cancer in patients previously operated on for T1-T2 N-M0 breast cancer. During the trial, blood samples were collected at baseline and on a yearly basis from most of the patients. Evaluation of drug and retinol concentrations by HPLC assay has been performed for all the samples to obtain 4-HPR pharmacokinetic information as well as information on the effect of 4-HPR in lowering retinol plasma levels. The most important criteria for validation and quality control of the HPLC assay are summarized in order to provide a guide and practical recommendations for analytical procedures to be performed during prevention trials. Studies have been performed on subsets of patients participating in the trial in order to identify circulating biomarkers predictive of breast cancer. Evidence has been obtained on a lowering effect of 4-HPR on biologically active IGF-I only in premenopausal women. This was due to a decrease of IGF-I, associated with a trend to an increase in IGF-I binding protein 3 (IGFBP-3). An interim analysis of the ongoing trial indicates that 4-HPR reduces the incidence of contralateral breast cancer only in premenopausal women. Analyses of total and unbound IGF-I are being performed on plasma samples collected at baseline and during intervention from women < or = 50 years old. The relationship between the incidence of a second breast cancer and the changes in IGF-I plasma levels will be assessed in order to validate IGF-I as a surrogate end point of contralateral breast cancer. The preliminary results of other studies on the effects of 4-HPR on tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), and urokinase plasminogen activator (uk-PA) and on the relevance of circulating p53 antibodies with relapse will be also presented.

Chemoprevention of breast cancer: the Italian experience.

The activity of our group is focused on the conduction of chemoprevention clinical trials of breast cancer in at-risk subjects, among which we include women on hormone replacement therapy (HRT). The role of the insulin-like growth factor (IGF) system and of mammographic breast density as surrogate biomarkers for breast cancer prevention is also being investigated. The IGF system is involved in human carcinogenesis of several solid tumors. IGF-I is a potent mitogen for breast cancer cells; elevated circulating IGF-I levels have been associated with a higher risk of premenopausal breast cancer, prostate and colorectal cancer in prospective studies. Both tamoxifen and the synthetic retinoid fenretinide (4-HPR) have been shown to decrease plasma IGF-I levels. A trial of their combination is ongoing in premenopausal women with increased risk for breast cancer. Mammographic breast density has also been associated with an increased risk of breast cancer in several prospective studies. In this article, we discuss the rationale for selection of appropriate cohorts, candidate agents, and putative surrogate biomarkers in our breast cancer prevention trials. Moreover, updated results of the secondary prevention trial of 4-H PR and of the primary prevention trial of tamoxifen are presented. Finally, the rationale for a reduction of tamoxifen dose in future prevention trials is provided.

Randomized trial of fenretinide to prevent second breast malignancy in women with early breast cancer.

BACKGROUND: Fenretinide, a vitamin A analogue, has been shown to inhibit breast carcinogenesis in preclinical studies. We determined the efficacy of fenretinide in preventing a second breast malignancy in women with breast cancer. METHODS: We randomly assigned 2972 women, aged 30-70 years, with surgically removed stage I breast cancer or ductal carcinoma in situ to receive for 5 years either fenretinide orally (200 mg/day) or no treatment. The primary end point was the incidence of contralateral breast cancer or ipsilateral breast cancer 7 years after randomization. Other end points considered post hoc were the same outcomes stratified by menopausal status, incidence of distant metastases, overall mortality, and tumors in other organs. The hazards of breast cancer occurrence were determined by Cox proportional hazards regression analysis. Statistical tests were two-sided. RESULTS: At a median observation time of 97 months, there were no statistically significant differences in the occurrence of contralateral breast cancer (P =.642) or ipsilateral breast cancer (P =.177) between the two arms. However, an interaction was detected between fenretinide treatment and menopausal status in both outcomes (P for interaction in both outcomes =.045), with a possible beneficial effect in premenopausal women (contralateral breast cancer: adjusted hazard ratio [HR] = 0.66, and 95% confidence interval [CI] = 0.41-1.07; ipsilateral breast cancer: adjusted HR = 0.65, and 95% CI = 0.46-0. 92) and an opposite effect in postmenopausal women (contralateral breast cancer: adjusted HR = 1.32, and 95% CI = 0.82-2.15; ipsilateral breast cancer: adjusted HR = 1.19, and 95% CI = 0.75-1. 89). There were no statistically significant differences between the two arms in tumors in other organs, incidence of distant metastasis, and all-cause mortality. CONCLUSIONS: Fenretinide treatment of women with breast cancer for 5 years appears to have no statistically significant effect on the incidence of second breast malignancies overall, although a possible benefit was detected in premenopausal women. These studies, particularly the post hoc analyses, are considered exploratory and need to be confirmed.

Role of retinoic acid receptor overexpression in sensitivity to fenretinide and tumorigenicity of human ovarian carcinoma cells.

The role of retinoic acid receptor (RAR) expression in sensitivity to N-(4-hydroxyphenyl)retinamide (4HPR or fenretinide) as well as on the tumorigenicity of human ovarian carcinoma cells was examined. Two human ovarian cancer cell lines, A2780 and IGROV-1, with a 10-fold difference in sensitivity to 4HPR were chosen to study RAR involvement in the response to 4HPR. To determine which RAR was effective, RARalpha, beta and gamma were individually overexpressed in A2780 cells, which are the most sensitive to 4HPR. Sensitivity to 4HPR was increased in RARbeta-overexpressing clones, whereas it was slightly decreased in RARalpha transfectants (which had diminished RARbeta expression) and was unchanged in clones transfected with RARgamma. IGROV-1 cells, which are RARbeta negative, were transfected with RARbeta. Surprisingly, none of the obtained IGROV-1 RARbeta transfectants expressed RARbeta protein, in spite of RARbeta mRNA transcription. All clones were similar to the parental IGROV-1 cells in their sensitivity to 4HPR. Treatment with a pharmacologically achievable concentration of 4HPR (1 microM) led to a rapid 2-fold increase in RARbeta mRNA levels in A2780 cells, but it did not induce RARbeta expression in IGROV-1 cells. Analysis of the tumorigenicity of A2780-transfected clones revealed that overexpression of RARalpha was associated with a significant reduction in tumor takes (50% and 67%, respectively, vs. 96% for the parent line) and with a reduced growth rate. Oncogenicity was clearly decreased in only 1 of the 2 RARbeta-overexpressing clones (33% takes) and was unchanged in the 2 clones with increased RARgamma expression. Our results demonstrate that basal expression and 4HPR inducibility of RARbeta play a role in mediating 4HPR response in ovarian cancer cells. The findings of reduced oncogenicity of clones overexpressing RARalpha and of one clone overexpressing RARbeta indicate that RARalpha and RARbeta might have a tumor-suppressive effect in ovarian tumors.

In vivo eradication of human BCR/ABL-positive leukemia cells with an ABL kinase inhibitor.

BACKGROUND: The leukemia cells of approximately 95% of patients with chronic myeloid leukemia and 30%-50% of adult patients with acute lymphoblastic leukemia express the Bcr/Abl oncoprotein, which is the product of a fusion gene created by a chromosomal translocation [(9:22) (q34;q11)]. This oncoprotein expresses a constitutive tyrosine kinase activity that is crucial for its cellular transforming activity. In this study, we evaluated the antineoplastic activity of CGP57148B, which is a competitive inhibitor of the Bcr/Abl tyrosine kinase. METHODS: Nude mice were given an injection of the Bcr/Abl-positive human leukemia cell lines KU812 or MC3. Tumor-bearing mice were treated intraperitoneally or orally with CGP57148B according to three different schedules. In vitro drug wash-out experiments and in vivo molecular pharmacokinetic experiments were performed to optimize the in vivo treatment schedule. RESULTS: Treatment schedules administering CGP57148B once or twice per day produced some inhibition of tumor growth, but no tumor-bearing mouse was cured. A single administration of CGP57148B caused substantial (>50%) but short-lived (2-5 hours) inhibition of Bcr/Abl kinase activity. On the basis of the results from in vitro wash-out experiments, 20-21 hours was defined as the duration of continuous exposure needed to block cell proliferation and to induce apoptosis in these two leukemia cell lines. A treatment regimen assuring the continuous block of the Bcr/Abl phosphorylating activity that was administered over an 11-day period cured 87%-100% of treated mice. CONCLUSION: These data indicate that the continuous block of the oncogenic tyrosine kinase of Bcr/Abl protein is needed to produce important biologic effects in vivo.

Increased retinol binding protein in the sera of patients with severe ischemic damage of the liver after transplantation.

OBJECTIVE: To study retinol binding protein variation in the serum of patients who have undergone liver transplantation. METHODS: Retinol binding protein was retrospectively determined by the immunonephelometric method on serum from 14 patients who had undergone orthotopic liver transplantation 2 weeks after the surgery and then once a month during the first year posttransplantation. The patients were divided into two groups on the basis of early (first 10 days) postoperative graft function: group I, 6 patients with severe ischemic damage; and II 8 patients with moderate-severe liver dysfunction. RESULTS: The men retinol binding protein level at one year of follow-up was persistently higher in group I than in group II (83.1 +/- 33.4 vs 44.6 +/- 20.7 mg/L, p < 0.001). Interestingly, retinol binding protein levels remained higher in patients of group I event when the other biochemical parameter of liver function returned to normal. The increase in retinol binding protein serum levels was independent of variation in other parameters of liver and kidney function, but was correlated with an increase in transthyretin and retinol levels. CONCLUSION: Our results show a close relationship between a permanent high retinol binding protein level and severe graft injury after liver transplantation. However, the mechanism underlying the increase remains to be defined.