A first-in-class, first-in-human, phase I trial of p28, a non-HDM2-mediated peptide inhibitor of p53 ubiquitination in patients with advanced solid tumours.

BACKGROUND: This first-in-human, phase I clinical trial of p28 (NSC745104), a 28-amino-acid fragment of the cupredoxin azurin, investigated the safety, tolerability, pharmacokinetics and preliminary activity of p28 in patients with p53(+) metastatic solid tumours. METHODS: A total of 15 patients were administered p28 i.v. as a short infusion three times per week for 4 weeks followed by a 2-week rest under an accelerated titration 3+3 dose escalation design until either a grade 3-related adverse event occurred or the maximum tolerated dose (MTD) was reached. Single-dose and steady-state serum pharmacokinetics were characterised. Assessments included toxicity, best objective response by RECIST 1.1 Criteria, and overall survival. RESULTS: No patients exhibited any dose-limiting toxicities (DLTs), significant adverse events or exhibited an immune response (IgG) to the peptide. The No Observed Adverse Effect Level (NOAEL) and MTD were not reached. Seven patients demonstrated stable disease for 7-61 weeks, three a partial response for 44-125 weeks, and one a complete response for 139 weeks. Three patients are still alive at 158, 140, and 110 weeks post therapy completion. CONCLUSION: p28 was tolerated with no significant adverse events. An MTD was not reached. Evidence of anti-tumour activity indicates a highly favourable therapeutic index and demonstrates proof of concept for this new class of non-HDM2-mediated peptide inhibitors of p53 ubiquitination.

The use of daunomycin-antibody immunoconjugates in managing soft tissue sarcomas: nude mouse xenograft model.

Analysis of human fibrosarcoma cells exposed to radiolabeled monoclonal antibody 19-24, which recognizes sarcoma-associated antigen p102, revealed that over 54% of the cell surface-bound radioactivity was internalized. No modulation of cell surface p102 antigen by monoclonal antibody 19-24 was observed in human fibrosarcoma cells. Monoclonal antibody 19-24 coupled to daunomycin via a dextran bridge was found to be most effective. In different preparations, the daunomycin:total protein molar ratio ranged from 1.9 to 6.1. In vitro cytotoxicity studies using human fibrosarcoma cells showed that, at 10 micrograms/ml concentration, this immunoconjugate was 79.4% as efficient as free daunomycin and, at 1 microgram/ml concentration, 36.8% as efficient. Control nonspecific murine monoclonal antibody P3 immunoconjugates were relatively ineffective. The distribution of 14C-Adriamycin, 125I-labeled monoclonal antibody 19-24, and 125I-labeled 19-24 immunoconjugate was also evaluated over a 24-h period in tumor and normal tissues of athymic mice bearing a human fibrosarcoma xenograft. Poor uptake of radiolabeled Adriamycin by the tumor tissue was observed. The level of 14C radioactivity in the tumor tissue never exceeded 1% of the total injected dose and was 24.8-fold lower than the radioactivity found in the spleen tissue. Tumor tissue uptake of radiolabeled monoclonal antibody 19-24 was characterized by the high tumor tissue:blood ratio of 1.62 +/- 0.28 (SD). However, for monoclonal antibody 19-24 immunoconjugates, this ratio decreased to 0.66 +/- 0.05, which was still higher than normal (liver, 0.48 +/- 0.02; lung, 0.48 +/- 0.07; spleen, 0.28 +/- 0.01) or nonspecific monoclonal antibody P3 immunoconjugates (0.22 +/- 0.03). Thus, it appears that, compared to free daunomycin, monoclonal antibody 19-24 immunoconjugates may be more efficient and less cytotoxic to normal tissues.

Effects of glucocorticoids on the growth of human fibrosarcoma cell line HT-1080.

The human fibrosarcoma cell line HT-1080 exhibits rapid growth following s.c. inoculation in 4-6-week-old male athymic mice. Cytosols from tumors carried in athymic mice bind glucocorticoid (Kd, 1.8 +/- 0.48 X 10(-8) M; Bmax, 240.5 +/- 35.3 fmol/mg cytosol protein, mean +/- SEM). Receptor sediments primarily in the 8-9S region on 5-20% sucrose gradients and is specific for the glucocorticoids. HT-1080 growth in vitro (as measured by cell count) was inhibited over a range of 10(-6)-10(-8) M after 7 days of incubation with dexamethasone and triamcinolone acetonide. Progesterone, estradiol, and dihydrotestosterone had no effect on HT-1080 growth in vitro. Preincubation with a 100-fold excess of progesterone reversed the growth inhibition observed with triamcinolone acetonide but not dexamethasone acetate. HT-1080 tumor cell growth responded biphasically to dexamethasone in vivo. Athymic mice given s.c. injections every other day with 5 or 25 micrograms dexamethasone showed an increase in tumor size inversely proportional to dose. In contrast, 200 micrograms of dexamethasone significantly inhibited tumor growth. Adrenalectomy did not significantly alter HT-1080 growth or glucocorticoid binding to tumor cytosols (Kd, 3.4 X 10(-8) +/- 1.1, Bmax, 236.9 +/- 9.9 fmol/mg cytosol protein, mean +/- SEM) although tumor incidence was decreased in sham adrenalectomized mice. Glucocorticoid binding in tumors grown in vivo was decreased by increasing amounts of dexamethasone. High pharmacological doses of glucocorticoids inhibit the growth of human fibrosarcomas in vivo and in vitro.

Histochemical and ultrastructural study of lactic dehydrogenase in chemically induced lung cancer.

Light and electron microscopy studies of lactic dehydrogenase activity were carried out in embryonic, neonatal, and adult mouse lungs and in lungs undergoing chemically induced carcinogenesis. Embryonic mouse lungs were collected on the 6th, 12th, and 18th days of gestation; 1-day-old lungs were used for the neonatal model. These were compared with adult normal mouse lung and lungs of the animals treated with 4-nitroquinoline 1-oxide at a monthly interval until cancer developed. Enzymatic activity was seen in the embryonic, precancerous, and malignant lung tissues and was found diffusely in the cytoplasm of the epithelial cells.

Biological behavior of MM1 hamster melanoma.

We have reported evidence recently for a high-affinity receptor for glucocorticoid Malignant Melanoma No. 1 hamster melanoma and suggested that tumor growth was facilitated by adrenal steroids. This report characterizes the behavior of Malignant Melanoma No. 1 following manipulation of the pituitary-adrenal axis in vivo. Bilateral adrenalectomy significantly retarded tumor growth. Hypophysectomy also significantly reduced tumor growth. Silastic implants of hydrocortisone in intact hamsters produced a dose (7 to 28 micrograms/day)-related increase in tumor growth. Implants releasing a low dose (3 micrograms/day) of dexamethasone also increased tumor growth. Chronic exposure of adrenalectomized and intact hamsters to a high dose (125 micrograms/day) of desoxycorticosterone acetate also produced a significant increase over adrenalectomized and sham-adrenalectomized controls. In contrast, chronic administration of adrenocorticotropic hormone and alpha-melanocyte-stimulating hormone to intact hamsters did not significantly alter melanoma growth. These observations support the suggestion that adrenocorticosteroids influence the growth of Malignant Melanoma No. 1 hamster melanoma and provide a model for studying the regulation of growth of a glucocorticoid-positive neoplasm originating outside the reticuloendothelial system.

Influence of the estrous cycle during carcinogen exposure on nitrosomethylurea-induced rat mammary carcinoma.

The influence of individual stages of the rat estrous cycle during exposure to I-nitroso-I-methylurea (NMU) on mammary tumor incidence, latency, number, and cytosol receptor dynamics for estrogen and progesterone was determined. Virgin female Buffalo rats were separated into three groups on Day 53 according to their vaginal smear pattern. NMU (5 mg/100 g body weight, i.v.) was administered in three monthly doses beginning at 53 to 55 days of age on diestrus, proestrus, or estrus between 9:00 and 11:00 a.m. Groups of rats had their second and third injections of NMU on the same day of the estrous cycle as their initial injection. All animals were killed during the morning on a diestrus day. Receptors for estrogen and progesterone were determined by a modified dextran-coated charcoal method and by sucrose density gradient analysis. Mean latencies to first tumor appearance in diestrus, proestrus, and estrus groups were 104.4, 83.6, and 91.4 days, respectively (p less than 0.05, diestrus versus estrus and proestrus) following the first NMU injection. The mean number of tumors per rat was significantly higher in rats injected on proestrus (4.5) or estrus (4.3) than on diestrus (2.0). Estradiol bound to receptor sedimented at 8 and 4 s and was suppressed by diethylstilbestrol and estradiol. Progesterone receptor migrated to 7.8 and 4 s regions. Estrogen receptor incidence (100%) and content (16.7 fmol/mg cytosol protein) was highest in rats injected on diestrus. In the proestrus and estrus injected groups, estrogen receptor incidence was 95 and 63% and content was 10.2 and 11.2 fmol/mg protein, respectively. The affinity of estradiol for its receptor was not significantly altered in any group. Although there were no statistically significant difference in progesterone receptor incidence or affinity between groups, progesterone receptor content (74.6 fmol/mg cytosol protein) was significantly higher in tumors from rats injected on proestrus than on diestrus. These data suggest that the prevailing hormonal milieu of the estrous cycle during NMU exposure may be critically important to the subsequent biological behavior and steroid receptor status of carcinogen-induced rat mammary tumors.

Presence of steroid receptors in human soft tissue sarcomas of diverse histological origin.

The incidence of cytosol receptors for androgen, estrogen, glucocorticoid, and progesterone was determined in a series of histologically diverse soft tissue sarcomas. Receptor binding was characterized in tumor specimens from 29 adult patients (14 female, 15 male) obtained during surgery. Nineteen (66%) tumor cytosols bound at least one steroid. Seven (24%) had more than one receptor. Ten of 23 (43%) cytosols assayed for androgen binding were positive (Kd 0.97 +/- 0.23 X 10(-9) M; 60.6 +/- 25.9 fmol/mg protein), 7 of 29 (24%) for estrogen (Kd 1.00 +/- 0.19 X 10(-9) M; 33.2 +/- 10.9 fmol/mg protein), 9 of 26 (35%) bound glucocorticoid (Kd 4.22 +/- 1.77 X 10(-9) M; 231.0 +/- 155.0 fmol/mg protein), and 1 of 28 (4%) bound progesterone (Kd 0.41 X 10(-9) M; 18.7 fmol/mg protein). Density gradient analysis suggested that androgen and estrogen binding was located predominantly in the 6S and 7 to 8S regions, respectively, whereas receptor for glucocorticoid sedimented at 8S. Steroid binding was not related to patient age. Estrogen-positive (71%) and glucocorticoid-positive (78%) cytosols appeared predominantly in tumors from female patients. Receptor distribution also appeared to be correlated to tumor histogenetic origin.

Monoclonal antibody identification and characterization of two human sarcoma-associated antigens.

Two murine monoclonal antibodies, 29-13 (IgG1) and 29-2 (IgG2a), generated against malignant fibrous histiocytoma plasma membranes immunoprecipitated a Mr 200,000 protein (p200), with an isoelectric point between 6.3 and 7.5. Two additional antibodies, 35-16 (IgG1) and 30-40 (IgG2a), generated against Ewing's sarcoma membranes, immunoprecipitated an acidic protein of Mr 160,000 (p160), with an isoelectric point between 5.8 and 6.7. Monoclonal antibodies 29-13 and 29-2 recognize a similar determinant(s) on p200 while 35-16 and 30-40 recognize different determinants on p160. Monoclonal antibody 29-13 exhibited significant binding to membranes isolated from fibrosarcoma and aggressive fibromatosis; moderate binding to osteosarcoma, hemangiopericytoma, and malignant fibrous histiocytoma; and minimal to no binding to other soft tissue sarcoma plasma membranes. The p200 protein was not expressed in 16 other malignant tumors and in only 3 of 35 normal human tissue specimens. High levels of p200 were selectively expressed by leiomyosarcoma, Ewing's sarcoma, and fibrosarcoma cells as well as neonatal fibroblasts in vitro, but not by other carcinoma cell lines or B-lymphoblasts. The p160 protein appeared to be selectively expressed by Ewing's sarcoma with little or no expression on other sarcomas, carcinomas, or normal tissues. However, the p160 antigen was expressed in Ewing's sarcoma, leiomyosarcoma, melanoma, 4 of 9 carcinomas, and neonatal fibroblasts in vitro. The affinity of MoAbs 29-13, 29-2, 35-16, and 30-40 ranged from 5.3 x 10(8) to 4.7 x 10(9) M-1 for sarcoma membranes with approximately 5 x 10(4) binding sites/sarcoma cell.

Effect of 17 beta-estradiol on the growth of estrogen receptor-positive human melanoma in vitro and in athymic mice.

Physiochemical properties of an estrogen binding protein were characterized in three human melanoma cell lines, UISO-MEL-1, UISO-MEL-2, and UISO-MEL-4. Estrogen binding to melanoma cytosol was saturable, specific for estrogens, and represented by a single class of high-affinity, limited-capacity binding sites (Kd 5.5 x 10(-10) M, 2.7 +/- 0.5 fmol/mg of cytosol protein, UISO-MEL-2; 2.2 x 10(-10) M, 7.8 +/- 3.3, UISO-MEL-4) (SEM). UISO-MEL-1 cytosols did not bind estradiol. The binding protein in UISO-MEL-2 and -4 sedimented at 8.5S and 9.2S, respectively, in the presence of 10 mM sodium molybdate. Solid-phase radioimmunoassay with a monoclonal antibody specific for human estrogen receptor (H222 sp lambda) showed good correlation (r = 0.84) with a hydroxyapatite biochemical assay of identical melanoma cytosols. Exposure of UISO-MEL-2 to estradiol produced a time- and temperature-dependent increase in total nuclear receptor for estrogen in vitro. Estradiol treatment of athymic mice also significantly increased cytosol progesterone receptor content in UISO-MEL-2 and UISO-MEL-4 xenografts. Estradiol had no effect on the plating efficiency or growth of any melanoma cell line or normal melanocytes in vitro. Tamoxifen also had no effect on melanoma growth in vitro. In contrast, chronic exposure of athymic mice carrying estrogen receptor-positive UISO-MEL-2 to estradiol resulted in a sex-dependent increase in tumor latency and overall inhibition of tumor growth. Taken together, these observations suggest that a subset of human melanomas contains limited amounts of an estrogen binding protein similar to that observed in other estrogen-responsive tissues. The lack of effect of estradiol on melanocyte and melanoma growth in vitro, coupled with a decrease in tumor growth in athymic mice, suggests that, while inhibition may be receptor mediated, possible indirect actions of estradiol must also be considered.

Pharmacokinetics of melphalan in clinical isolation perfusion of the extremities.

The pharmacokinetics of melphalan in clinical hyperthermic isolation perfusion was studied in 16 patients with malignant melanoma. Analysis by computer-generated lines of best fit showed that the loss of melphalan from perfusate conforms best to a biexponential equation. The initial loss with a half-life (t1/2) of approximately 5 to 10 min is interpreted as rapid uptake of melphalan by the tissue of the perfused extremity. The terminal portion of the curve with a half-life of approximately 35 to 50 min is interpreted as due predominantly to the hydrolysis of melphalan, with a lesser component of loss due to absorption of melphalan to the filters and tubing of the perfusion apparatus. Determination of the area under the curve suggests that there is no appreciable uptake of melphalan by the tissue of the perfused extremity after 30 min.

Micropthalmia transcription factor: a new prognostic marker in intermediate-thickness cutaneous malignant melanoma.

Micropthalmia transcription factor (Mitf) is involved in melanocyte development and differentiation. The current study was undertaken to determine whether there is a relationship between Mitf expression and survival in patients with intermediate-thickness (1.0-4.0 mm) melanoma. Original paraffin blocks or slides of the primary tumor were accessible in 63 such patients. Mitf expression was evaluated by immunocytochemistry and analyzed visually. Slides were graded as follows according to the percentage of cells whose nuclei stained positive for Mitf: (a) 0, 0%; (b) +1, 1-25%; (c) +2, 26-50%; (d) +3, 51-75%; and (e) +4, > 75%. Median follow-up was 50 months. Mean thickness was 2.2 +/- 0.7 mm. Mean overall survival was 171.90 +/- 13.12 months. Mean disease-free survival was 168.53 +/- 13.96 months. Fifty-two melanomas (82.5%) stained positive for Mitf. By univariate analysis, mean overall survival and disease-free survival in patients whose melanomas did not express Mitf were 80.89 +/- 17.98 months (median, 51 months) and 71.36 +/- 19.87 months (median, 40 months), respectively. This compares with 187.90 +/- 13.41 months (median, not reached) and 186.78 +/- 13.84 months (median, not reached), respectively, for patients whose melanomas expressed Mitf (P = 0.0086 and P = 0.0054). These findings persisted in multivariate analysis. In addition, patients with > 50% Mitf expression had significantly fewer nodal metastases after node dissection than patients with < or = 50% Mitf expression (P = 0.04). Our data suggest that Mitf may be a new molecular prognostic marker in patients with intermediate-thickness melanoma.

Differential proliferative response to linoleate in cultures of epithelial cells from normal human breast and fibroadenomas.

Human breast epithelial cells isolated from normal breast tissues of premenopausal women demonstrated direct evidence of a proliferative effect by linoleate (18:2 omega 6) or prostaglandin E2 (PGE2) in the presence of insulin and epidermal growth factor in serum-free cultures within a collagen gel matrix. Neither epidermal growth factor nor 18:2 omega 6 by itself was capable of stimulating growth but together they stimulated proliferation synergistically. Epithelial cells isolated from fibroadenomas on the other hand failed to exhibit any growth stimulation due to 18:2 omega 6 or PGE2. The linoleate-stimulated growth in normal breast epithelial cells was inhibited by indomethacin, a cyclooxygenase inhibitor, which however could be reversed by PGE2. The proliferative response of normal breast epithelial cells to 18:2 omega 6 was accompanied by a greater conversion of [14C]18:2 omega 6 to arachidonic acid and [14C]20:4 omega 6 to prostaglandins than that seen in epithelial cells from fibroadenomas. The turnover of [14C]18:2 omega 6 in the phospholipids of normal cells was higher than in fibroadenomas indicating a possible role of phospholipids in mediating the 18:2 omega 6 effect in normal cells. Both normal and fibroadenoma cells can proliferate in response to cholera toxin and glucocorticoids when supplemented to the insulin- and epidermal growth factor-containing medium. From the results it appears that, unlike normal cells, fibroadenoma cells may have a specific defect in the PGE2-responsive cyclic AMP-generating mechanism whereas cholera toxin-induced mechanism is operative in both types of cells.

Estrogen receptor in malignant melanoma.

The significance of an estrogen binding protein (ER) in malignant melanoma remains controversial. We have prospectively assayed for ER on 141 patients with malignant melanoma and correlated the presence of the ER with known prognostic variables. The overall incidence of ER was 43%. The incidence of ER in males was 38.7% and 50% in females (not significant). There is an increased incidence of ER+ melanoma in women with extremity lesions (P = .08). The disease-free interval (DFI), survival, and recurrent interval were 42.0 +/- 4.0, 52.3 +/- 4.3, 13.7 +/- 1.7 months in ER- patients; 63.7 +/- 11.6, 76.1 +/- 11.4, 26.5 +/- 7.3 months in ER+ patients (1 to 10 fmol/mg cytosol protein), and 69.8 +/- 17.9, 102.7 +/- 27.9, 29.4 +/- 9.9 months in ER+ patients (greater than 10 fmol/mg cytosol); respectively. When ER+ groups were combined, the DFI in women with ER+ lesions was significantly longer than those with ER- tumors (P less than .05). Cox multivariate analysis demonstrated that ER status is a significant variable of survival along with thickness level and nodal status. These observations suggest that ER may be a marker for a more biologically indolent melanoma.

Evaluation of newer prognostic markers for adult soft tissue sarcomas.

PURPOSE: In addition to tumor size, grade, location, and the presence of metastases, other factors may be useful in prognostication for adults with soft tissue sarcoma (STS). This study examines the relationship of MDR-1 mRNA, p-glycoprotein (P-gp), Ki-67 expression, and DNA content expression to clinical outcome in adults with STS. PATIENTS AND METHODS: Snap-frozen STS specimens from 65 patients were analyzed and compared with clinical outcomes. Immunohistochemistry was performed for the Ki-67 antigen and P-gp. DNA content was determined using the Feulgen reaction and quantitated using image analysis. MDR-1 mRNA expression was determined using a reverse-transcriptase polymerase chain reaction (RT-PCR)-based assay. RESULTS: P-glycoprotein expression was found by immunohistochemistry in 48% of cases with 5-year overall (54% v 14%, P = .07) and disease-free survival rates (32% v 18%, P = .039) higher in high-grade tumors that did not express P-gp. MDR-1 mRNA was detected in 51% of cases and no patient with high levels of MDR-1 mRNA expression was a long-term survivor. Patients with diploid tumors had significantly better survival than those with nondiploid tumors (51% v 31%, P = .03). High levels of Ki-67 were associated with poorer overall survival (46% v 31%, P = .04). On multivariate analysis, American Joint Committee on Cancer (AJCC) staging, DNA content, Ki-67, and P-gp staining were significant prognostic factors for 5-year overall and disease-free survival. CONCLUSION: P-gp expression, high-level Ki-67 expression, and nondiploid DNA content are independent prognostic indicators that correlate with poor outcomes in STS patients. However, MDR-1 mRNA was not found to be predictive of survival. These newer markers are useful additions to AJCC staging for prognostication for patients with STS. Such markers may be useful in selecting high-risk STS patients who could benefit from systemic adjuvant therapy.

Plasma c-erbB-2 levels in breast cancer patients: prognostic significance in predicting response to chemotherapy.

PURPOSE: To determine the significance of plasma c-erbB-2 levels to assess the extent of disease spread and to predict the response to chemotherapy in node-positive breast cancer patients. METHODS: We determined plasma levels of c-erbB-2 in 79 stages II and III breast cancer patients who received cyclophosphamide, methotrexate, and flourouracil (CMF)/cyclophosphamide, methotrexate, fluorouracil, vincristine, and prednisone (CMFVP) chemotherapy. All patients had a minimum follow-up of greater than 60 months or until disease recurrence. Plasma samples were obtained before and after chemotherapy. Plasma c-erbB-2 levels were quantified by enzyme-linked immunoassay. c-erbB-2 levels were analyzed in relation to the patients' axillary lymph node status, menopausal status, disease status, disease-free survival (DFS), and steroid receptor status of tumor. RESULTS: Plasma c-erbB-2 levels varied widely in breast cancer patients. In general, when all patients were included in the analyses, plasma c-erbB-2 levels before chemotherapy correlated significantly with the number of positive axillary lymph nodes and with postchemotherapy c-erbB-2 levels. No association was observed between pre- or postchemotherapy c-erbB-2 levels and other variables (patients' age at diagnosis, receptor status of the tumor, or disease status). The prognostic significance of different factors (ie, nodal status [one to three v > three positive nodes], menopausal status [pre- v postmenopausal women], estrogen receptor [ER] status [ER+ v ER-], and pre- and postchemotherapy c-erbB-2 levels) in predicting DFS was determined in all study patients. Among the variables examined, nodal status was the strongest predictor of DFS in these patients. The second most significant prognostic marker was postchemotherapy c-erbB-2 level. Prechemotherapy c-erbB-2 levels showed prognostic significance for DFS in a subset of breast cancer patients (ie, patients with > three positive nodes). Patients with greater than three positive lymph nodes and those with greater than 100 fmol/mL of plasma c-erbB-2 levels before therapy had significantly shorter DFS than did those patients with 100 fmol/mL or less c-erbB-2 levels. CONCLUSION: In breast cancer patients, determination of c-erbB-2 levels before therapy is an important biomarker to assess the extent of disease spread in the lymph nodes. Postchemotherapy c-erbB-2 levels are also a prognostic indicator for DFS in patients who receive chemotherapy. Finally, in a subgroup of patients with greater than three positive nodes, prechemotherapy c-erbB-2 levels are a prognostic marker for response of patients to standard chemotherapy.

Resveratrol inhibits rhabdomyosarcoma cell proliferation.

Rhabdomysarcoma is the most common soft tissue tumour in children under the age of 15. Although the introduction of multimodal treatment programmes, including chemotherapy, radiation therapy and excision have increased the overall survival, the chemotherapeutic agents currently used for the treatment of rhabdomyosarcoma exhibit considerable toxicity. The aim of this study was to investigate the effects and possible mechanism(s) of action of resveratrol on human embryonal rhabdomyosarcoma (RD) cells. Resveratrol is a natural polyphenolic compound produced in a number of edible plants and has received considerable attention as a potential chemopreventive and/or chemotherapeutic agent against various types of cancers. In the present study, resveratrol was shown to inhibit cell proliferation of RD cells in a dose-dependent manner with an IC50 of 48.1 micromol/l and induce an arrest in the S/G2 phase of the cell cycle. As evident from immunocytochemical data, resveratrol treatment increased the size of the RD cells. Furthermore, resveratrol treatment resulted in a significant downregulation of cyclin B expression as demonstrated by western blot analyses. In conclusion, the present study shows that resveratrol exerts a strong inhibition of rhabdomyosarcoma cell proliferation in part by arresting cells in S/G2 phase of the cell cycle. These findings warrant further investigation to establish potential use of resveratrol as a relatively non-toxic chemotherapeutic agent for the treatment of rhabdomyosarcoma.

Mutant p53 expression: a marker of diminished survival in well-differentiated soft tissue sarcoma.

The aim of this study was to assess the translational value of the quantitative assay of mutant p53 protein expression as both a prognostic indicator and a tool to determine appropriate therapy in a group of relatively innocuous and morphologically similar soft tissue sarcomas (STSs). Using a quantitative ELISA, we analyzed mutant p53 protein expression in 47 well-differentiated (grade I) STSs from patients treated in our Department of Surgical Oncology. Sixteen of 47 tumors expressed up to 42.6 ng mutant p53 protein/mg total protein. After a mean follow-up of 112 months, 63% of the patients with mutant p53+ tumors but only 16% of the patients with mutant p53- tumors had died (P < 0.01). Mutant p53 expression of >/=4.5 ng predicted even greater reduction in survival. These data show that mutant p53 expression identifies biologically aggressive grade I STSs. This molecular marker should have translational value as a tool to select those patients likely to benefit from aggressive multimodal therapy and intense surveillance.

Phase I evaluation of interleukin-2-transfected irradiated allogeneic melanoma for the treatment of metastatic melanoma: appendix 1: protocol.

A cell line (UISO-H-MEL-2) was established from the neoplastic cells of a patient with malignant melanoma during the natural course of the patient's treatment. The melanoma cells express defined MHC Class I histocompatibility determinants including determinants specified by the HLA-A2 Class I allele, along with a common melanoma-associated T-cell epitope derived from the tyrosinase gene. The gene for human interleukin-2 (IL-2) was transduced into the cells with a provirus (pZipNeoSVIL-2), packaged in GP + envAM12 cells. Integration of the IL-2 gene into genomic DNA of the transduced cells and its expression were established. The IL-2-secreting cell line (UISO-H-MEL-2-IL-2) was found to be free of recombinant retroviruses and other infectious agents. The IL-2-secreting cells will be subjected to 5000 rads X-irradiation and administered to 12 informed patients with metastatic malignant melanoma in a Phase I toxicity study. The dose of X-irradiation was sufficient to inactivate one hundred percent of the cells, but insufficient to completely inhibit IL-2 synthesis during a fourteen-day period of analysis. Patients who have failed all standard forms of treatment will become eligible for inclusion in the study if they develop metastatic melanoma, and if their tumor cells express products of the tyrosinase gene. The patients will differ with the cellular immunogen at no less than three of six MHC Class I alleles, but will share identity at the HLA-A2 Class I allele. The patient's antimelanoma immune response to the injected cells will be determined by both in vivo and in vitro parameters. Background studies performed in inbred mice indicate that X-irradiated IL-2-secreting cells that express both melanoma-associated antigens and allogeneic Class I histocompatibility antigens are more antigenic in terms of their capacity to induce an antimelanoma response than X-irradiated IL-2-secreting melanoma cells. Of significance for the future potential of this form of therapy in melanoma patients, the period of survival of mice was established melanoma treated with the IL-2-secreting allogeneic cells was significantly (P < 0.001) longer than that of untreated animals, or animals treated with X-irradiated melanoma cells. An analogous protocol was reviewed and approved by the Recombinant DNA Advisory Committee of the National Institutes of Health.

Photoperiodic control of melanoma growth in hamsters: influence of pinealectomy and melatonin.

Pinealectomy (PX) increased MM1 (melanotic melanoma no. 1) hamster melanoma growth in animals held under a 14-h light, 10-h dark (14:10) photoperiod without altering tumor latency. Hamsters maintained under a 6-h light, 18-h dark (6:18) photoperiod exhibited gonadal collapse, a longer tumor latency, and slower tumor growth rate than animals held under 14:10. PX produced a further increase in tumor latency and a decrease in growth in these animals. In contrast, acute morning injection of low doses (50 micrograms/day) of melatonin or delivery by Silastic capsule (35 micrograms/day) implanted at the time of tumor cell inoculation increased MM1 melanoma growth in hamsters held under 14:10 photocycle, without affecting testicular or adrenal function. Treatment of hamsters 11 weeks before tumor cell inoculation with 14 micrograms/day melatonin via Silastic capsule produced a decrease in serum PRL but no change in tumor growth or testicular or adrenal weights in animals held under 14:10. Treatment of hamsters with 17.7 micrograms/day melatonin (Silastic capsule) 11 weeks before tumor cell inoculation increased testes and adrenal weights as well as serum PRL and androgen levels, but significantly decreased tumor growth in hamsters held under a short daily photoperiod. These results suggest that the photoperiod under which hamsters are maintained dictates the growth rate of MM1 tumors and the effect of PX on tumor behavior. When photoperiod significantly alters gonadal and adrenal function, the quantity, time, and duration of melatonin presentation are all important variables in the effect of melatonin on tumor growth.

Induction of differentiation by 1alpha-hydroxyvitamin D(5) in T47D human breast cancer cells and its interaction with vitamin D receptors.

The role of the active metabolite of vitamin D, 1,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), in cell differentiation is well established. However, its use as a differentiating agent in a clinical setting is precluded due to its hypercalcaemic activity. Recently, we synthesised a relatively non-calcaemic analogue of vitamin D(5), 1alpha-hydroxyvitamin D(5) (1alpha(OH)D(5)), which inhibited the development of carcinogen-induced mammary lesions in culture and suppressed the incidence of chemically induced mammary carcinogmas in rats. In the present study, we determined the differentiating effects of 1alpha-(OH)D(5) in T47D human breast cancer cells and compared its effects with 1,25(OH)(2)D(3). Cells incubated with either 10 or 100 nM of the analogues inhibited cell proliferation in a dose-dependent manner, as measured by the dimethylthiazolyl-2,5-diphenyltetrazolium bromide (MTT) assay. Similar growth-inhibitory effects were also observed for MCF10(neo) cells. Both vitamin D analogues induced cell differentiation, as determined by induction of casein expression and lipid production. However, MCF10(neo) cells failed to respond to either vitamin D analogue and did not undergo cell differentiation. Since the cell differentiating effect of vitamin D is considered to be mediated via the vitamin D receptor (VDR), we examined the induction of VDR using reverse transcriptase-polymerase chain reaction (RT-PCR) in both cells. The results showed that, in T47D cells, both 1,25(OH)(2)D(3) and 1alpha(OH)D(5) induced VDR in a dose-dependent manner. Moreover, both analogues of vitamin D upregulated the expression of vitamin D response element-chloramphenicol acetyl transferase (VDRE-CAT). These results collectively indicate that 1alpha-(OH)D(5) may mediate its cell-differentiating action via VDR in a manner similar to that of 1,25(OH)(2)D(3).