Inhibition of angiogenesis and induction of endothelial and tumor cell apoptosis by green tea in animal models of human high-grade non-Hodgkin's lymphoma.

Recent reports suggest that green tea consumption may prevent or delay the growth of human cancer, possibly by impairing tumor invasion and/or by an anti-angiogenic effect. In NOD/SCID mice transplanted intraperitoneally with human non-Hodgkin's lymphoma (NHL) cell lines, Namalwa, RAP1-EIO and HS-Sultan, green tea prevented 50% of Namalwa tumors (P = 0.0017 by log-rank) and significantly inhibited RAP1-EIO and HS-Sultan tumor growth. Notably, treatment with the chemotherapy drug cyclophosphamide at the maximum tolerable dose was unable to prevent Namalwa tumor occurrence. In the three models evaluated, the frequency of apoptotic endothelial and tumor cells was significantly increased in mice given green tea compared to controls. These results support further trials in NHL to evaluate whether green tea, alone or in combination with chemotherapy, may delay or prevent disease progression.

Endostatin, an antiangiogenic drug, induces tumor stabilization after chemotherapy or anti-CD20 therapy in a NOD/SCID mouse model of human high-grade non-Hodgkin lymphoma.

Both chemotherapy and chimeric anti-CD20 monoclonal antibodies are effective agents against B-cell non-Hodgkin lymphoma (NHL). However, patients achieving remission are at risk of relapse. To evaluate the effect of the antiangiogenic drug endostatin used alone and after the administration of cyclophosphamide (CTX) or the anti-CD20 antibody rituximab, we generated a new model of human NHL by transplanting Namalwa cells intraperitoneally into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. First, we determined the most effective treatment schedule for the drugs assessed. When administered alone, CTX (3 courses of 75 mg/kg of body weight given intraperitoneally), rituximab (3 courses of 25 mg/kg given intraperitoneally), and endostatin (5 courses of 50 microg given subcutaneously) delayed tumor growth, and CTX was the most effective in controlling bulky disease. When given after chemotherapy or immunotherapy, endostatin effectively induced tumor stabilization. When mice given CTX or rituximab on days 3, 5, and 7 after transplantation were randomly assigned to receive endostatin or phosphate-buffered saline on days 15 to 19, tumor growth was prevented in endostatin-treated mice as long as the drug was administered. Furthermore, administration of endostatin on days 25 to 29 after tumor regrowth still induced significant tumor regression, whereas CTX and rituximab were not effective. The specific antiangiogenic action of endostatin was confirmed by in vitro and in vivo studies indicating that the drug inhibited proliferation and induced apoptosis of endothelial (but not of NHL) cells. In conclusion, sequential administration of chemotherapy and endostatin seems promising for treating bulky NHL, and the less toxic sequential administration of rituximab and endostatin is promising for treating limited disease. (

Human myeloid and lymphoid malignancies in the non-obese diabetic/severe combined immunodeficiency mouse model: frequency of apoptotic cells in solid tumors and efficiency and speed of engraftment correlate with vascular endothelial growth factor production.

Recent studies have suggested that non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice transplanted with human hematological malignancies show higher levels of engraftment compared with other strains. We used this model to compare xenotransplantability of human leukemia and lymphoma cell lines and to investigate angiogenesis in hematopoietic malignancies. Ten of 12 evaluated cell lines were able to engraft NOD/SCID mice within 120 days. A strong correlation was observed between the amount of vascular endothelial growth factor (VEGF) produced in vitro by cultured cells and the efficiency of tumor engraftment (r = 0.808; P = 0.001), and an inverse correlation was found between VEGF production and the time of tumor engraftment (r = -0.792; P = 0.006) and between VEGF production and the frequency of apoptotic/dead cells in solid tumors (r = -0.892; P = 0.007). Moreover, VEGF production correlated with the frequency of endothelial (CD31+/CD34+) cells in solid tumors (r = 0.897; P = 0.001). Taken together with in vitro data presented here and indicating that the VEGF antagonist Flt-1/Fc chimera inhibits leukemia and lymphoma cell proliferation, our findings support a role for tumor-derived VEGF in leukemia and lymphoma progression. Furthermore, the present study confirms previous observations indicating that VEGF expression may play a crucial role in xenotransplantability of human solid malignancies in SCID mice. The NOD/SCID model is promising for future evaluations of antiangiogenic drugs, alone or in combination with established chemo- or immunotherapy regimens.

The Gly571Arg mutation, associated with the autonomic and sensory disorder congenital insensitivity to pain with anhidrosis, causes the inactivation of the NTRK1/nerve growth factor receptor.

Point mutations affecting the NTRK1/TRKA gene, encoding one of the receptors for the nerve growth factor (NGF), have been detected in congenital insensitivity to pain with anhidrosis (CIPA), a human hereditary sensory neuropathy characterized by absence of reaction to noxious stimuli and anhidrosis. To define the defect of NTRK1 in CIPA patients, we have introduced one of the previously reported mutations (Gly571Arg) into both the NTRK1 and the TRK-T3 oncogene cDNAs. The expression of the mutated constructs into COS1 cells revealed that the introduced mutation, while not affecting its correct membrane localization, rendered the NTRK1 protein unable to undergo activation upon stimulation with NGF. Similarly, the mutation abolished the constitutive activation of the TRK-T3 oncogene. Transfection into NIH3T3 and PC12 cells showed the loss of transforming and differentiating activity by the mutated constructs. Our results demonstrate clearly that the CIPA mutations cause the inactivation of the NTRK1 receptor, thus exerting a loss of function effect, and provide an experimental approach to distinguish functional mutations from genetic polymorphisms.

Transforming activity of the chimeric sequence formed by the fusion of collagen gene COL1A1 and the platelet derived growth factor b-chain gene in dermatofibrosarcoma protuberans.

As a consequence of a reciprocal translocation t(17;22)(q22;q13) and of supernumerary ring chromosomes derived from the t(17;22), a fusion between the platelet-derived growth factor b-chain (PDGF, c-sis proto-oncogene) and the collagen type 1A1 (COL1A1) genes has been recently described in dermatofibrosarcoma protuberans (DP), an infiltrating skin tumor (Simon et al., 1997). Although PDGFB has been implicated in transforming processes via autocrine and paracrine pathways, by the activation of the cognate receptor, no direct evidence of its involvement in neoplastic transformation of human tumours has been so far provided. In this report, we have tested the DNA from four DPs in the classical DNA transfection assay onto NIH3T3 fibroblast cell line. All the DNAs induced the formation of transformed foci in the transfected cultures whose derived cell lines were shown to contain a fused sequence comprising the human COL1A1 and PDGF genes. The relative breakpoint regions have been sequenced revealing that this gene fusion deleted exon 1 of PDGF and released the growth factor from its normal regulation. All the biochemical and biological assays were consistent with the model of an autocrine mechanism for NIH3T3 transformation by the human rearranged PDGFB gene involving the activation of the endogenous PDGF receptor.

Role of the TFG N-terminus and coiled-coil domain in the transforming activity of the thyroid TRK-T3 oncogene.

The thyroid TRK-T3 oncogene results from the fusion of the tyrosine kinase (TK) domain of NTRK1 (one of the receptors for the Nerve Growth Factor) on chromosome 1 to sequences of a novel gene, TFG, on chromosome 3. The 68 kDa TRK-T3 fusion oncoprotein displays a constitutive tyrosine kinase activity resulting in its capability to transform mouse NIH3T3 cells. The TFG portion of TRK-T3 contains a coiled-coil domain most likely responsible for the constitutive, ligand-independent activation of the receptor tyrosine kinase activity. We have previously shown that TRK-T3 oncoprotein forms, in vivo, complexes of three or four molecules. By mean of different experimental approaches, we show here that TRK-T3 activity depends on oligomers formation. In addition, the analysis of different TRK-T3 mutants indicates that the TFG coiled-coil domain and its N-terminal region are both required for the activation and the fully transforming activity of the TRK-T3 oncoprotein, although, most likely, they play a role in different steps of the transforming process. The deletion of the coiled-coil domain abrogates the oligomers formation leading to a constitutive activation; the deletion of the N-terminal region, although not affecting phosphorylation and complexes formation, abrogates transformation, thus suggesting a role in cellular localization and/or interaction with substrata.

Chromosome 1 rearrangements involving the genes TPR and NTRK1 produce structurally different thyroid-specific TRK oncogenes.

The NTRK1 gene in the q arm of chromosome 1 encodes one of the receptors for the nerve growth factor and is frequently activated as an oncogene in papillary thyroid carcinomas. The activation is due to chromosomal rearrangements juxtaposing the NTRK1 tyrosine kinase domain to 5'-end sequences from different genes. The thyroid TRK oncogenes are activated by recombination with at least three different genes: the gene coding for tropomyosin and TPR, both on chromosome 1,and TFG on chromosome 3. In a previous study, we showed that two tumors carrying the TPR/NTRK1 rearrangement contained structurally different oncogenes named TRK-T1 and TRK-T2. In this paper, we report (1) the cDNA structure of TRK-T2, (2) evidence that TRK-T2 is generated by different rearrangements in two thyroid tumors, and (3) a detailed analysis of the three different TPR/NTRK1 rearrangements. With molecular studies based on Southern blot hybridization, cloning, and sequencing, we show that all the rearrangements are nearly balanced, involving deletion, insertion, or duplication of only few nucleotides. In one case, an additional rearrangement involving sequences derived from chromosome 17 was detected.

Replacement of Fhit in cancer cells suppresses tumorigenicity.

The candidate tumor suppressor gene, FHIT, encompasses the common human chromosomal fragile site at 3p14.2, the hereditary renal cancer translocation breakpoint, and cancer cell homozygous deletions. Fhit hydrolyzes dinucleotide 5',5"'-P1,P3-triphosphate in vitro and mutation of a central histidine abolishes hydrolase activity. To study Fhit function, wild-type and mutant FHIT genes were transfected into cancer cell lines that lacked endogenous Fhit. No consistent effect of exogenous Fhit on growth in culture was observed, but Fhit and hydrolase "dead" Fhit mutant proteins suppressed tumorigenicity in nude mice, indicating that 5',5"'-P1, P3-triphosphate hydrolysis is not required for tumor suppression.

Hypocholesterolemic effect of yogurt and milk.

In order to determine the effect of milk products on serum cholesterol, triglycerides, and diet, 54 volunteers were studied for varying periods with dietary supplementation of nonpasteurized yogurt, pasteurized yogurt and 2% butterfat milk. Serum cholesterol was significantly reduced by 5 to 10% after 1 week of supplementation with either nonpasteurized or pasteurized yogurt; 2% butterfat milk reduced serum cholesterol to a smaller and less significant effect. Serum triglycerides were unaffected by the diet and dietary intake studies confirmed that intake of other nutrients remained relatively stable throughout the study. Supplementation of diet with yogurt may have a helpful hypocholesterolemic effect.