Biomedical subjects

Massimo Tommasino

Publications and source records attributed to Massimo Tommasino.

At least 19 recordsLinked to original sources

PubMed2006

NKIM-6, a new immortalized human brain capillary endothelial cell line with conserved endothelial characteristics.

Primary human brain capillary endothelial cells (hBCECs) are available only in small quantities and have a short life span in vitro; this restricts their use as in vitro model for the blood-brain barrier (BBB). To overcome these limitations, we have established an immortalized hBCEC line (NKIM-6) by transfection with pLXSN16E6E7, which encodes the human papillomavirus type 16 E6 and E7 genes. The cell line exhibits an extended life span in vitro and retains its characteristic endothelial morphology, endothelial markers, and physiology. Likewise, as demonstrated by immunohistochemistry and reverse transcription/polymerase chain reaction (RT-PCR), NKIM-6 cells express BBB markers, and the lack of glial, neuronal, and epithelial markers confirms their endothelial origin. Moreover, with quantitative RT-PCR, we have been able to demonstrate that several ATP-binding cassette-transporters are expressed in NKIM-6 cells with a conserved expression order compared with primary hBCECs. Our results suggest that this cell line might be suitable as in vitro model for several aspects of the BBB.

ATP-Binding Cassette Transporters↗

PubMed2006

Tumor suppressive activity of a variant isoform of manganese superoxide dismutase released by a human liposarcoma cell line.

A cell line derived from a pleiomorphic liposarcoma, named LSA, was previously reported to secrete (a) factor(s) exhibiting oncotoxic properties. The present article describes the isolation, purification and sequence analysis of a protein released by LSA cells into conditioned culture medium. This protein proved to be a variant isoform of manganese superoxide dismutase (MnSOD), hence its designation as LSA-type-MnSOD. This LSA-type-SOD differed from conventional SODs in its secretion by producer cells, contrasting with the normal localization of SODs in the mitochondrial matrix. Interestingly, during the protein purification process, LSA-type-SOD cosegregated with a cytotoxic activity directed against a number of tumor cell lines, as determined under in vitro conditions. This cytopathic effect was most likely due to LSA-type-SOD, since it could be fully reproduced using recombinant SOD that was expressed from cDNA clones isolated from LSA cells mRNA preparations and henceforth designated L-rSOD. In addition to its manifestation in cell lines kept in tissue culture, the oncotoxicity of LSA-type-SOD was further reflected in a remarkable capacity of this protein for suppression of mammary tumors in Balb-C-FR(III) mice. Animals subcutaneously injected with L-rSOD in the tumor area showed a complete disruption of established mammary carcinomas, as monitored by nuclear magnetic resonance (NMR) scanning. Moreover, metastatic spreading, which was readily detected in the control group, was suppressed in the treated animals. Altogether these data suggest that LSA-type-SOD interferes with survival and spreading of neoplastically transformed cells and deserves to be future validated as a therapeutic agent against cancer, either alone or in combination with conventional treatments.

Amino Acid Sequence↗

PubMed2006

Human papillomavirus type 77 E7 protein is a weak deregulator of cell cycle.

Human Papillomavirus type 77 is a skin type found in non-melanoma skin cancers of immuno-compromised individuals. Although, the HPV77 E6 oncoprotein has been well studied, nothing is known about E7. Studies on mucosal HPV types (e.g. HPV16) showed that E7 deregulates the cell cycle by binding to and promoting degradation of retinoblastoma protein (pRb). Here, we characterized the impact of HPV77 E7 on the cell cycle. We observed that HPV77 E7 associated with pRb with a lower affinity than HPV16 E7, promoting weakly its degradation. Although, HPV16 E7 led to cellular proliferation and accumulation of the cell cycle inhibitor p16(INK4a), both events were not clearly observed in HPV77 E7 cells. Together, these data indicate that HPV77 E7 does not efficiently deregulate the cell cycle, in contrast to several E7s of mucosal HPV types.

Animals↗

PubMed2006

HPV16 E6 natural variants exhibit different activities in functional assays relevant to the carcinogenic potential of E6.

Genetic studies have revealed natural amino acid variations within the human papillomavirus (HPV) type 16 E6 oncoprotein. To address the functional significance of E6 polymorphisms, 10 HPV16 E6 variants isolated from cervical lesions of Swedish women were evaluated for their activities in different in vitro and in vivo assays relevant to the carcinogenic potential of E6. Small differences between E6 prototype and variants, and among variants, were observed in transient expression assays that assessed p53 degradation, Bax degradation, and inhibition of p53 transactivation. More variable levels of activities were exhibited by the E6 proteins in assays that evaluated binding to the E6-binding protein (E6BP) or the human discs large protein (hDlg). The E6 prototype expressed moderate to high activity in the above assays. The L83V polymorphism, previously associated with risk for cancer progression in some populations, expressed similar levels of activity as that of the E6 prototype in most functional assays. On the other hand, L83V displayed more efficient degradation of Bax and binding to E6BP, but lower binding to hDlg. Results of this study indicate that naturally occurring amino acid variations in HPV16 E6 can alter activities of the protein important for its carcinogenic potential.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Skin human papillomavirus type 38 alters p53 functions by accumulation of deltaNp73.

The E6 and E7 of the cutaneous human papillomavirus (HPV) type 38 immortalize primary human keratinocytes, an event normally associated with the inactivation of pathways controlled by the tumour suppressor p53. Here, we show for the first time that HPV38 alters p53 functions. Expression of HPV38 E6 and E7 in human keratinocytes or in the skin of transgenic mice induces stabilization of wild-type p53. This selectively activates the transcription of deltaNp73, an isoform of the p53-related protein p73, which in turn inhibits the capacity of p53 to induce the transcription of genes involved in growth suppression and apoptosis. DeltaNp73 downregulation by an antisense oligonucleotide leads to transcriptional re-activation of p53-regulated genes and apoptosis. Our findings illustrate a novel mechanism of the alteration of p53 function that is mediated by a cutaneous HPV type and support the role of HPV38 and deltaNp73 in human carcinogenesis.

Animals↗

PubMed2006

HPV16 variants in squamous intraepithelial lesions in human immunodeficiency virus-negative and -positive Brazilian women.

Emerging evidence supports the role of human papillomavirus (HPV) intratype variations in the development of cervical lesions in immunocompetent women, but few studies investigated HPV16 variants in human immunodeficiency virus (HIV)-positive women. This is the first study in Brazil evaluating HPV16 variants in women with (n = 19) and without (n = 22) HIV infection, as well as cervical lesions. Although non-European variants presented an almost 3-fold increase (13.6% vs. 36.8%) among HIV-positive women, associations between HPV16 variants and HIV infection did not reach statistical significance (Fisher's exact test, p = 0.15). No associations were found between non-European variants and HSIL (Fisher's exact test, p = 0.41), although a significant association was observed between European variants and HSIL among HIV-negative women (Fisher's exact test, p = 0.01). In conclusion, in HIV-negative women the HPV16 European variants seem to influence the occurrence of HSIL, whereas in HIV-positive women, similar roles are atributted to both variants.

Adolescent↗

PubMed2006

Development of a sensitive and specific assay combining multiplex PCR and DNA microarray primer extension to detect high-risk mucosal human papillomavirus types.

The importance of assays for the detection and typing of human papillomaviruses (HPVs) in clinical and epidemiological studies has been well demonstrated. Several accurate methods for HPV detection and typing have been developed. However, comparative studies showed that several assays have different sensitivities for the detection of specific HPV types, particularly in the case of multiple infections. Here, we describe a novel one-shot method for the detection and typing of 19 mucosal high-risk (HR) HPV types (types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, and 82). This assay combines two different techniques: multiplex PCR with HPV type-specific primers for amplification of viral DNA and array primer extension (APEX) for typing. This novel method has been validated with artificial mixtures of HPV DNAs and clinical samples that were already analyzed for the presence of mucosal HPV types by a different consensus PCR method, i.e., GP5+/GP6+. Our data showed a very good agreement between the results from the multiplex PCR/APEX assay and those from the GP5+/GP6+ PCR (overall rates of HPV positivity, 63.0 and 60.9%, respectively). Whereas the GP5+/GP6+ PCR was slightly more sensitive for the detection of HPV type 16 (HPV-16), multiplex PCR-APEX found a higher number of infections with HPV-33, HPV-53, and multiple HPV types. These favorable features and the high-throughput potential make our present novel assay ideal for large-scale clinical and epidemiological studies aimed at determining the spectrum of mucosal HR HPV types in cervical specimens.

DNA Primers↗

PubMed2006

A nuclear export signal and phosphorylation regulate Dok1 subcellular localization and functions.

Dok1 is believed to be a mainly cytoplasmic adaptor protein which down-regulates mitogen-activated protein kinase activation, inhibits cell proliferation and transformation, and promotes cell spreading and cell migration. Here we show that Dok1 shuttles between the nucleus and cytoplasm. Treatment of cells with leptomycin B (LMB), a specific inhibitor of the nuclear export signal (NES)-dependent receptor CRM1, causes nuclear accumulation of Dok1. We have identified a functional NES (348LLKAKLTDPKED359) that plays a major role in the cytoplasmic localization of Dok1. Src-induced tyrosine phosphorylation prevented the LMB-mediated nuclear accumulation of Dok1. Dok1 cytoplasmic localization is also dependent on IKKbeta. Serum starvation or maintaining cells in suspension favor Dok1 nuclear localization, while serum stimulation, exposure to growth factor, or cell adhesion to a substrate induce cytoplasmic localization. Functionally, nuclear NES-mutant Dok1 had impaired ability to inhibit cell proliferation and to promote cell spreading and cell motility. Taken together, our results provide the first evidence that Dok1 transits through the nucleus and is actively exported into the cytoplasm by the CRM1 nuclear export system. Nuclear export modulated by external stimuli and phosphorylation may be a mechanism by which Dok1 is maintained in the cytoplasm and membrane, thus regulating its signaling functions.

Active Transport, Cell Nucleus↗

PubMed2006

Increased risk for cervical disease progression of French women infected with the human papillomavirus type 16 E6-350G variant.

To test the significance of human papillomavirus (HPV) type 16 and HPV16 E6 variants as risk factors for viral persistence and progression to high-grade lesion, we did a nested case-control study within a cohort study of >15,000 Caucasian French women. Three groups infected with high-risk HPV were compared: (a) women with cleared infection (controls, n = 201), (b) women with persistent infection (cases, n = 87), and (c) women who progressed into high-grade lesion (cases, n = 58). Women with persistent HPV infection and those that progressed into high-grade lesions were likelier to harbor HPV16 than other high-risk HPV types [odds ratio (OR), 2.4; 95% confidence interval (95% CI), 1.3-4.3 and OR, 4.2; 95% CI, 2.2-8.1, respectively]. Notably, especially elevated ORs of persistence (3.0; 95% CI, 1.4-6.7) and progression (6.2; 95% CI, 2.7-14.3) were found among women who harbored the HPV16 350G variant. Thus, HPV type and HPV16 variant seem to be risk factors for viral persistence and progression of infections into high-grade cervical lesions.

Case-Control Studies↗

PubMed2005

Dok1 expression and mutation in Burkitt's lymphoma cell lines.

Dok1 is an adaptor tyrosine kinase substrate with tumor-suppressive activity. The gene encoding Dok1 maps to human chromosome 2p13, which is frequently rearranged in human tumors. We have previously reported a frameshift mutation of this gene and the down-regulation of its expression in chronic lymphocytic leukemia. In this study, we have determined the expression levels of Dok1 in Burkitt's lymphoma (BL) cell lines, lymphoblastoid cell lines from patients with X-linked lymphoproliferative (XLP-LCL), or from control healthy donors. We have also screened for Dok1 gene mutations by heteroduplex analysis and direct sequencing. Dok1 expression was down-regulated in all BL and XLP-LCL cell lines in comparison to the control cells. No Dok1 mutation or polymorphism was found in the coding region of Dok1 in the three types of cells. However, DNA sequence analysis revealed the presence of four nucleotide changes in Dok1 gene, T(90172)C (intron 1), C(89487)T and (89433)InsCTCT (intron 2), and A(87714)G (3' UTR). T(90172)C and (89433)InsCTCT that were detected in about 7% of BL, 9% of XLP-LCL and 4% of normal samples may represent a common polymorphism. C(89487)T and A(87714)G changes were detected in 9 and 6% of analyzed BL lines, respectively, but never in the control and XLP-LCL cells, indicating that these nucleotide substitution occurred during tumor development. Interestingly, the C(89487)T variant is associated with a significantly lower level of Dok1 expression compared to the control samples. A positive association was also found between the presence of EBV in BL and the Dok1 genetic variation. Our data show that Dok1 expression and structure are affected in a subset of Burkitt's lymphoma samples, suggesting its possible role in this type of cancer.

Base Sequence↗

PubMed2005

Papillomavirus infection in the conjunctiva of individuals with and without AIDS: an autopsy series from Uganda.

HIV and genus beta human papillomavirus (HPV) types have been associated with squamous cell carcinoma of the conjunctiva (SCC). To determine whether conjunctival HPV infection is associated with AIDS, we analysed 136 lesion-free eye biopsies and tested for genera alpha, beta and gamma HPV types. Only infections with genera beta and gamma HPV types was found. After adjustment for age and gender, no excess of genera beta or gamma HPV infection was found in individuals who had died of or with AIDS compared to those who had died of other infectious diseases [relative risk (RR)=1.3; 95% confidence interval (CI): 0.4-4.8], or chronic diseases or trauma (RR=0.9; 95% CI: 0.3-2.9). Our findings suggest that infection with genera beta or gamma HPV types in lesion-free conjunctivas is common, but not greatly enhanced by the presence of AIDS.

Acquired Immunodeficiency Syndrome↗

PubMed2005

Skin hyperproliferation and susceptibility to chemical carcinogenesis in transgenic mice expressing E6 and E7 of human papillomavirus type 38.

The oncoproteins E6 and E7 of human papillomavirus type 38 (HPV38) display several transforming activities in vitro, including immortalization of primary human keratinocytes. To evaluate the oncogenic activities of the viral proteins in an in vivo model, we generated transgenic mice expressing HPV38 E6 and E7 under the control of the bovine homologue of the human keratin 10 (K10) promoter. Two distinct lines of HPV38 E6/E7-expressing transgenic mice that express the viral genes at different levels were obtained. In both lines, HPV38 E6 and E7 induced cellular proliferation, hyperplasia, and dysplasia in the epidermis. The rate of occurrence of these events was proportional to the levels of HPV38 E6 and E7 expression in the two transgenic lines. Exposure of the epidermis of nontransgenic mice to UV led to p21WAF1 accumulation and cell cycle arrest. In contrast, keratinocytes from transgenic mice continued to proliferate and were not positive for p21WAF1, indicating that cell cycle checkpoints are altered in keratinocytes expressing the viral genes. Although the HPV38 E6/E7-expressing transgenic mice did not develop spontaneous tumors during their life span, two-stage carcinogen treatment led to a high incidence of papillomas, keratoacanthomas, and squamous-cell carcinomas in HPV38 mice compared with nontransgenic animals. Together, these data show that HPV38 E6 and E7 display transforming properties in vivo, providing further support for the role of HPV38 in carcinogenesis.

Animals↗

PubMed2005

Analysis of E7/Rb associations.

The product of the early gene E7 is one of the major transforming proteins of human papillomaviruses (HPVs). It exerts its activity by associating with and altering the biological functions of several cellular proteins involved in the control of fundamental events, such as cell proliferation and apoptosis. The best-characterized activity of E7 from HPV type 16, the most frequently detected type in cervical cancer, is its ability to bind and induce degradation of the tumor-suppressor retinoblastoma protein (pRb) via the ubiquitin pathway. pRb plays a key role in cell-cycle control by negatively regulating, via direct association, the activity of several transcription factors, including members of the E2F family. The neutralization of pRb functions mediated by E7 results in constitutive activation of the transcription factors, with consequent loss of cell-cycle control. Several studies have shown that the oncogenic potential of a specific HPV type is dependent on the efficiency of E7 in targeting pRb. In this chapter, we describe two methods to measure the efficiency of the E7 proteins from different HPV types in neutralizing the pRb functions. The first one, the plate-binding assay, allows the determination of the pRb binding affinity of E7 proteins, while the second one permits the analysis of their impact on the pRb pathway in intact cells.

3T3 Cells↗

PubMed2004

The Na+-H+ exchanger-1 induces cytoskeletal changes involving reciprocal RhoA and Rac1 signaling, resulting in motility and invasion in MDA-MB-435 cells.

INTRODUCTION: An increasing body of evidence shows that the tumour microenvironment is essential in driving neoplastic progression. The low serum component of this microenvironment stimulates motility/invasion in human breast cancer cells via activation of the Na+-H+ exchanger (NHE) isoform 1, but the signal transduction systems that underlie this process are still poorly understood. We undertook the present study to elucidate the role and pattern of regulation by the Rho GTPases of this serum deprivation-dependent activation of both NHE1 and subsequent invasive characteristics, such as pseudopodia and invadiopodia protrusion, directed cell motility and penetration of normal tissues. METHODS: The present study was performed in a well characterized human mammary epithelial cell line representing late stage metastatic progression, MDA-MB-435. The activity of RhoA and Rac1 was modified using their dominant negative and constitutively active mutants and the activity of NHE1, cell motility/invasion, F-actin content and cell shape were measured. RESULTS: We show for the first time that serum deprivation induces NHE1-dependent morphological and cytoskeletal changes in metastatic cells via a reciprocal interaction of RhoA and Rac1, resulting in increased chemotaxis and invasion. Deprivation changed cell shape by reducing the amount of F-actin and inducing the formation of leading edge pseudopodia. Serum deprivation inhibited RhoA activity and stimulated Rac1 activity. Rac1 and RhoA were antagonistic regulators of both basal and stimulated tumour cell NHE1 activity. The regulation of NHE1 activity by RhoA and Rac1 in both conditions was mediated by an alteration in intracellular proton affinity of the exchanger. Interestingly, the role of each of these G-proteins was reversed during serum deprivation; basal NHE1 activity was regulated positively by RhoA and negatively by Rac1, whereas RhoA negatively and Rac1 positively directed the stimulation of NHE1 during serum deprivation. Importantly, the same pattern of RhoA and Rac1 regulation found for NHE1 activity was observed in both basal and serum deprivation dependent increases in motility, invasion and actin cytoskeletal organization. CONCLUSION: Our findings suggest that the reported antagonistic roles of RhoA and Rac1 in cell motility/invasion and cytoskeletal organization may be due, in part, to their concerted action on NHE1 activity as a convergence point.

Actins↗

PubMed2004

p53 mutations are common in human papillomavirus type 38-positive non-melanoma skin cancers.

In cervical cells, the E6 protein of the oncogenic human papillomavirus (HPV) types inactivates p53, promoting its degradation. Consequently, mutations of the p53 gene are rarely seen in these cancers. Our recent data indicate that the cutaneous HPV38 is involved in skin carcinogenesis. In this study, we have determined the presence of HPV38 and the status of p53 gene in 32 non-melanoma skin cancers. We found that p53 gene is frequently mutated in HPV38-positive skin cancers and that HPV38 E6 does not promote p53 degradation. Thus, different mechanisms appear to be involved in the development of HPV-positive cervical and skin cancers.

Animals↗

PubMed2004

The short stature homeodomain protein SHOX induces cellular growth arrest and apoptosis and is expressed in human growth plate chondrocytes.

Mutations in the homeobox gene SHOX cause growth retardation and the skeletal abnormalities associated with Léri-Weill, Langer, and Turner syndromes. Little is known about the mechanism underlying these SHOX-related inherited disorders of bone formation. Here we demonstrate that SHOX expression in osteogenic stable cell lines, primary oral fibroblasts, and primary chondrocytes leads to cell cycle arrest and apoptosis. These events are associated with alterations in the expression of several cellular genes, including pRB, p53, and the cyclin kinase inhibitors p21(Cip1) and p27(Kip1). A SHOX mutant, such as seen in Léri-Weill syndrome patients, does not display these activities of the wild type protein. We have also shown that endogenous SHOX is mainly expressed in hypertrophic/apoptotic chondrocytes of the growth plate, strongly suggesting that the protein plays a direct role in regulating the differentiation of these cells. This study provides the first insight into the biological function of SHOX as regulator of cellular proliferation and viability and relates these cellular events to the phenotypic consequences of SHOX deficiency.

Antimetabolites, Antineoplastic↗

PubMed2004

High levels of the mitochondrial large ribosomal subunit protein 40 prevent loss of mitochondrial DNA in null mmf1 Saccharomyces cerevisiae cells.

Members of the YERO57c/YJGFc/UK114 protein family have been identified in bacteria and eukaryotes. The budding yeast Saccharomyces cerevisiae contains two different proteins of this family, Hmf1p and Mmf1p. We have previously shown that Mmf1p is a mitochondrial protein functionally related to its human homologue and able to influence the maintenance of mitochondrial DNA. Deletion of Mmf1 results in loss of the mitochondrial genome. Using a multicopy suppression approach, we have identified a protein of the mitochondrial large ribosomal subunit, MRPL40, which stabilizes mtDNA in Deltammf1 cells. Overexpression of MRPL40 did not prevent loss of mtDNA in a mutant strain lacking the mitochondrial protein Abf2p. Thus, MRPL40 does not have a general effect on mtDNA stability, but it may be specific for the mmf1-null strain. We also show that the Deltamrpl40 cells present a similar phenotype to the mmf1-null strain, having reduced mtDNA stability and growth rate. Furthermore, we observed that rho(+)Deltamrpl40 haploid cells can be obtained when tetrads are directly dissected on medium containing a non-fermentable carbon source. Thus, replication and segregation of the mtDNA can occur in the absence of MRPL40. We also show that another mitochondrial ribosomal protein, MRPL38, is able to overcome the Deltammf1-associated defect. Together, our results suggest a link between Mmf1p and the two mitochondrial ribosomal proteins.

Blotting, Western↗

PubMed2004

Generation of a DNA microarray for determination of E6 natural variants of human papillomavirus type 16.

Infection with high-risk types of human papillomavirus (HPV) is necessary for the development of cervical cancer. However, the majority of the HPV infections are efficiently cleared by the immune system and only a minority persist and induce the development of malignant lesions. Several studies provided evidence that intratype genetic variations are implicated in determining the clinical outcome of HPV infections. In this study, we describe a DNA chip based on arrayed primer extension (APEX) for the analysis of the natural variants of HPV16, the most frequently detected type in cervical cancer world-wide. We show that HPV16 E6 variants are detected efficiently by APEX. In addition, APEX is a valid assay for the simultaneous detection of different HPV16 variants. Thus, it could be used for clinical studies aiming to analyse multiple infections with different HPV16 subtypes. Finally, since APEX combines the advantages of dideoxy DNA sequencing with the high-throughput potential of the microarray, it is suitable for the simultaneous analysis of a number of variable regions of HPV16 genome in large-scale studies.

DNA, Viral↗