Maria Grazia Sacco

Early and multifocal tumors in breast, salivary, Harderian and epididymal tissues developed in MMTY-Neu transgenic mice

Transgenic mice carrying various oncogenes driven by mammary gland specific enhancers develop mammary tumors usually arising in a stochastic way. The only exception is a mouse lineage (TG.NF) carrying an activated rat Neu oncogene driven by the murine mammary tumor virus long terminal repeat (MMTV-LTR) that gave rise to rapid and multifocal mammary tumors interpreted as a result of a single-step neoplastic transformation. The effect of the oncogene appeared to be specific for breast tissue, since salivary and Harderian glands as well as epididymis expressed high levels of Neu but only developed hyperplasia (Muller et al., Cell, (1988) 54, p. 105). Here we describe a transgenic mouse lineage for the MMTV-Neu, analysed up to third generation. Multifocal tumors involving mammary glands arose very rapidly in all females independently from pregnancy and in some males. Moreover, multifocal neoplasias occurred also in salivary and Harderian glands and in the epididymis at a very high rate. These data demonstrate that the Neu oncogene can induce tumors in all the tissues where it is expressed at high levels.

Whole genome analysis and microRNAs regulation in HepG2 cells exposed to cadmium.

Cadmium (Cd) is a metal known to be toxic and carcinogenic, but its mechanism of action remains to be fully elucidated. We investigated the gene expression modulation in the human hepatoma cell line HepG2 after exposure to 2 μM and 10 μM Cd using an Agilent microarray. Furthermore, we evaluated the microRNA modulation after exposure to 10 μM Cd with a Low Density Array. At the low concentration only eleven genes belonging to the metallothionein familiy were regulated. At the higher concentration the pathway enrichment analysis for the 536 up-regulated genes showed a large number of pathways related to cancer, whereas the 424 down-regulated genes were enriched on pathways correlated to liver function. A large percentage of modified microRNAs belonged to the let-7 family, which is considered to have oncosuppressor functions. Several pathways connected to cancer were regulated at the transcription level, and miRNAs had a potential impact on the modulation of this regulation.

Systemic gene therapy with anti-angiogenic factors inhibits spontaneous breast tumor growth and metastasis in MMTVneu transgenic mice

Tumor growth and metastasis are angiogenesis-dependent. The possibility of inhibiting tumor growth by interfering with the formation of new vessels has recently raised considerable interest. We previously reported that it is possible to inhibit primary tumor growth and metastasis in a transgenic model of spontaneous breast tumor, which shows many similarities to its human counterpart (including ability to metastasize) by intratumoral administration of a DNA construct carrying the murine angiostatin cDNA driven by liposomes. Here we report that it is also possible to achieve this goal by a systemic (intraperitoneal) delivery of therapeutic DNA constructs carrying genes coding for mouse and human anti-angiogenic factors which include angiostatin, endostatin and TIMP-2. These findings may be relevant to the design of therapeutic interventions in humans.

Microenvironmental control of malignancy exerted by RNASET2, a widely conserved extracellular RNase

A recent body of evidence indicates an active role for stromal (mis)-regulation in the progression of neoplasias. Within this conceptual framework, genes belonging to the growing but still poorly characterized class of tumor antagonizing/malignancy suppressor genes (TAG/MSG) seem to play a crucial role in the regulation of the cross-talk between stromal and epithelial cells by controlling malignant growth in vivo without affecting any cancer-related phenotype in vitro. Here, we have functionally characterized the human RNASET2 gene, which encodes the first human member of the widespread Rh/T2/S family of extracellular RNases and was recently found to be down-regulated at the transcript level in several primary ovarian tumors or cell lines and in melanoma cell lines. Although we could not detect any activity for RNASET2 in several functional in vitro assays, a remarkable control of ovarian tumorigenesis could be detected in vivo. Moreover, the control of ovarian tumorigenesis mediated by this unique tumor suppressor gene occurs through modification of the cellular microenvironment and the induction of immunocompetent cells of the monocyte/macrophage lineage. Taken together, the data presented in this work strongly indicate RNASET2 as a previously unexplored member of the growing family of tumor-antagonizing genes.

Cell fusion is a physiological process in mouse liver.

A large portion of hepatocytes are polyploid cells, thought to arise through endoduplication followed by aborted cytokinesis. However, several recent reports describing liver cell fusion with exogenously derived bone marrow cells have been published. The exact significance of this finding is unclear, because the adopted protocols involve ablation regimens, damaged livers and artificial injections of adult cells. By creating chimeric mice bearing distinct reporter genes (LacZ and GFP), we show that in an unperturbed setting, hepatocytes carrying both markers can be detected via immunohistochemistry and polymerase chain reaction analysis. To further corroborate these findings with a direct visualization of the chromosome content at the single‐cell level, we performed genotype analysis via fluorescence in situ hybridization on XY/XX chimeric mice with a Y chromosome–specific paint and an X chromosome–specific bacterial artificial chromosome clone probes. Conclusion: This technique confirmed the occurrence of cell fusion in adult mouse liver. (HEPATOLOGY 2008.)

Lymphoid abnormalities in CD40 ligand transgenic mice suggest the need for tight regulation in gene therapy approaches to hyper immunoglobulin M (IgM) syndrome

Mutations in the CD40 ligand (CD40L) are responsible for human hyper immunoglobulin M (IgM) syndrome. The absence of the interaction between CD40L, expressed by T lymphocytes, and the CD40 receptor present on the surface of B cells is responsible for the inability of B cells to carry out the isotype switch from IgM to the other Ig classes. This leads to a fatal immunodeficiency for which no cure exists. For these reasons, the CD40L gene is a good candidate for gene therapy studies. To investigate the possible effects of the expression of this tightly regulated gene in vivo, we produced transgenic mice in which CD40L expression was deregulated. Widespread ectopic expression appears to be lethal. Overexpression in mature T cells is compatible with life, but in one-third of the cases, mice developed atypical lymphoid proliferations which, occasionally, progressed into frank lymphomas. Even though gene therapy is one of the most promising approaches to cure human hyper IgM syndrome, these results suggest that when we modify very tightly regulated genes such as cytokines or other growth factors, particular care has to be taken to avoid excessive stimulation of the target cells.

Establishment and characterization of a new mammary adenocarcinoma cell line derived from MMTV neu transgenic mice

A new murine cell line, named MG1361, was established from mammary adenocarcinomas arising in a MMTV-neu transgenic mouse lineage where breast tumors develop in 100% of females, due to the over-expression of the activated rat neu oncogene in the mammary gland. The MG1361 cell line shows an epithelial-like morphology, has a poor plating efficiency, low clonogenic capacity, and a doubling time of 23.8 hours. Karyotype and flow cytometry analysis revealed a hypotetraploid number of chromosomes, whereas cell cycle analysis showed 31.2% of cells to be in the G1 phase, 21.4% in S and 47.4% in G2 + M. This cell line maintains a high level of neu expression in vitro. The MG1361 cell line was tumorigenic when inoculated in immunodeficient (nude) mice and the derived tumors showed the same histological features as the primary tumors from which they were isolated. MG1361 cells were positive for specific ER and PgR binding which was competed by tamoxifen, making this cell line useful for the evaluation of endocrine therapy. Moreover, they were sensitive to etoposide treatment, suggesting that they could be a model for the study of chemotherapy-induced apoptosis. As the tumors arising in MMTV-neu transgenic mice have many features in common with human mammary adenocarcinomas (Sacco et al., Gene Therapy 1995; 2: 493–497), this cell line can be utilized to perform basic studies on the role of the neu oncogene in the maintenance of the transformed phenotype, and to test novel protocols of therapeutic strategies.

Assessment of an automated in vitro basal cytotoxicity test system based on metabolically-competent cells.

When in vitro test systems are evaluated for assessment of the toxicity of chemical compounds, particular efforts are made to mimic the in vivo reality as close as possible. Cellular models with appropriate metabolic competence, i.e. with the potency to biotransform chemical compounds, are considered crucial since some metabolites have a different toxicity than their parent compounds. In this study a cell based in vitro test system is proposed to investigate the basal cytotoxicity of several reference chemicals. Both metabolic competent HepaRG cells and cells with no or low hepatic enzyme activity (undifferentiated HepaRG and proliferating HepG2) were used. The classic Neutral Red Uptake (NRU) assay proved to be robust and reliable to be applied as viability assay. The test was performed on a robotic platform, which enabled fully automated and simultaneous screening of the compounds. The outcome of these tests grouped the tested compounds in three categories following their detoxification effect (benzo(a)pyrene, valproic acid), their bio-activation effect (aflatoxin B1) and their specific effect on inhibition of cell proliferation (cycloheximide, sodium lauryl sulphate, atropine sulphate monohydrate, acetylsalicylic acid).

Growth of human melanoma xenografts is suppressed by systemic angiostatin gene therapy.

The effect of local and systemic delivery of the angiostatin gene on human melanoma growth was studied in nude mice. Liposome-coated plasmids carrying the cDNA coding for murine and human angiostatin (CMVang and BSHang) were injected weekly, locally or systemically, in mice transplanted with melanoma cells. The treatment reduced melanoma growth by 50% to 90% compared to that occurring in control animals treated with liposome-coated plasmid carrying the lacZ gene or in untreated controls. The growth of both locally injected and controlateral uninjected tumors in mice bearing two melanoma grafts was significantly suppressed after intratumoral treatment. Tumor growth inhibition was also observed in mice treated by intraperitoneal delivery, suggesting that angiostatin gene therapy acts through a systemic effect. Both melanoma growth suppression and delay in the onset of tumor growth were observed in treated mice. PCR performed on tumors and normal tissues showed that the lipofected DNA was present in tissues from treated mice, and angiostatin expression was demonstrated by RT-PCR. Histopathological analysis of melanoma nodules revealed an increase in apoptotic cells and a reduction in vessel density in tumors from treated mice. Our results suggest that systemic, liposome-mediated administration of genes coding for antiangiogenic factors represents a promising strategy for melanoma treatment in humans. Cancer Gene Therapy (2001) 8, 491–496

Combined antiestrogen, antiangiogenic and anti-invasion therapy inhibits primary and metastatic tumor growth in the MMTVneu model of breast cancer.

Treatments available to women with locally advanced breast cancer are unsatisfactory, since most patients succumb to metastatic spread. Therefore, there is a need to devise novel therapeutic combinations that effectively inhibit metastatization and to test them in animal models of breast cancer showing strong similarities with their human counterpart, including the ability to give rise to metastases. With these considerations in mind, tamoxifen (TAM), 4-hydrotamoxifen (4-HT) or liposome-complexed DNA constructs coding for antiangiogenic/anti-invasion proteins (angiostatin, TIMP-2, IFN-α1, sFLT-1) were individually administered to MMTVneu transgenic mice. Significant inhibition of primary tumor growth was obtained with TAM (40% inhibition, P=0.049), angiostatin (85% inhibition, P=0.001) and TIMP-2 (60% inhibition, P=0.015). No lung metastasis was observed in any of these treated mice at 5 months, compared with a rate of 70% in control groups. These observations were the basis for designing a combined treatment with all these compounds. The association of angiostatin, TIMP-2 and TAM was greatly effective at the primary tumor level (90% inhibition, P=0.01). Moreover, all the mice treated with this association were metastasis free at a time point (6 months) in which seven out of nine control mice were either dead from disseminated cancer or showed lung metastasis. This combined therapy could become an important component of anticancer therapy in humans.