Mandibular bi-directional distraction osteogenesis: A technique to manage both transverse and sagittal mandibular diameters via a lingual tooth-borne acrylic plate and double-hinge bone anchorage.

PURPOSE: Mandibular hypoplasia can develop transversely, sagittally, or in both diameters simultaneously. Current techniques achieve either sagittal or transverse expansion with different surgeries. Here, we present a novel method to obtain transverse and sagittal mandibular distraction in one stage. MATERIALS AND METHODS: The technique consists of a double osteotomy: a dento-alveolar osteotomy comprising four or six anterior teeth and a vertical symphysiotomy underneath. The mandibular basal bone is immediately expanded transversely and fixed to the lower symphysis via a miniplate carrying only one screw on each side that functions as a hinge during active distraction. The plate is connected to the anterior dento-alveolar block with a metal wire ligature. A teeth-anchored lingual distraction system can expand transversely at the alveolar bone level and then sagittally with the anterior dento-alveolar segment wired to the lower plate. RESULTS: Satisfying and stable results were achieved, confirmed by measurements on serial plaster casts. CONCLUSION: To the best of our knowledge, this is the first proposal for ortho-surgical correction of both transversal and sagittal mandibular hypoplasia via a bi-directional distraction procedure. A combination of bone-hardware anchorage and dental-anchored distraction systems is suggested. Transmucosal hardware emergence and need for a second surgery to remove bone-borne appliances are avoided.

Ultrasound biomicroscopy in small animal research: applications in molecular and preclinical imaging.

Ultrasound biomicroscopy (UBM) is a noninvasive multimodality technique that allows high-resolution imaging in mice. It is affordable, widely available, and portable. When it is coupled to Doppler ultrasound with color and power Doppler, it can be used to quantify blood flow and to image microcirculation as well as the response of tumor blood supply to cancer therapy. Target contrast ultrasound combines ultrasound with novel molecular targeted contrast agent to assess biological processes at molecular level. UBM is useful to investigate the growth and differentiation of tumors as well as to detect early molecular expression of cancer-related biomarkers in vivo and to monitor the effects of cancer therapies. It can be also used to visualize the embryological development of mice in uterus or to examine their cardiovascular development. The availability of real-time imaging of mice anatomy allows performing aspiration procedures under ultrasound guidance as well as the microinjection of cells, viruses, or other agents into precise locations. This paper will describe some basic principles of high-resolution imaging equipment, and the most important applications in molecular and preclinical imaging in small animal research.

Targeted therapy for advanced prostate cancer: Looking through new lenses.

Prostate cancer is the most common noncutaneous malignancy in men and also the third leading cause of death due to cancer in males. The conventional initial therapy for localized advanced or metastatic disease is hormone or androgen deprivation therapy. Although hormone-based therapies generally result in rapid responses, the disease then progresses to a phase when they fail to control the malignancy despite castrate testosterone levels. Some patients with castration-resistant prostate cancer continue to respond to secondary hormonal manipulations, and docetaxel-based chemotherapy improves median survival to about 18 months. Prostate cancer is termed hormone-refractory when it no longer responds to hormonal therapy. Currently, other therapeutic options, such as radical prostatectomy, radiation therapy or cryotherapy offer improvement in survival mostly in early stages. New therapy approaches based on a deeper understanding of especially metastatic prostate cancer are of vital importance. Here we discuss up-to-date clinical trials of agents with novel targets and present paradigms in prostate cancer vaccine therapy, metastasis suppressor genes, and some provocative findings on combination therapies of cytotoxic agents, which might provide a platform for developing effective treatment for advanced prostate cancer.

A small molecule based on the pRb2/p130 spacer domain leads to inhibition of cdk2 activity, cell cycle arrest and tumor growth reduction in vivo.

One strategy in the development of anticancer therapeutics has been to arrest malignant proliferation through inhibition of the enzymatic activity of cyclin-dependent kinases (cdks), which are key regulatory molecules of the cell cycle. Over the past few years, numerous compounds with remarkable cdk inhibitory activity have been studied in cancer therapy, although it is very difficult to point out the best cdk to target. An excellent candidate appears to be cdk2, whose alteration is a pathogenic hallmark of tumorigenesis. The small molecule described in our study showed an inhibitory effect on the kinase activity of cdk2, a significant growth arrest observed in a colony formation assay and a reduction in the size of the tumor in nude mice, thus suggesting its potential role as a promising new type of mechanism-based antitumor drug, also for the treatment of hyperproliferative disorders.

Cell cycle control and beyond: emerging roles for the retinoblastoma gene family.

Rb family proteins (pRb/p105, Rb2/p130 and p107) play a key role in cell cycle control and are worthily involved in transcription repression and tumor suppression. The mechanisms of transcriptional activation and repression by the Rb gene family has been extensively investigated: pRb, pRb2/p130 and p107 interact with different E2F family factors and can inhibit E2F responsive promoters, interfering with progression of cell cycle, gene transcription, initiation of apoptotic process and cell differentiation. Recent studies have indicated that Rb and Rb2/p130 may be involved in cellular response to DNA damage events, by influencing the transcription of factors involved in DNA repair pathways. In particular, evidences suggest that Rb loss and target gene deregulation impacts on the repair of UV-induced pyrimidine pyrimidone photoproducts (6-4 PP) by regulating the expression of several DNA damage factors involved in UV DNA damage repair processes, including proliferating cell nuclear antigen. Ongoing studies are focused on the mechanisms by which Rb family genes drive cell cycle exit following DNA damage induction, and how Rb gene family's interaction with chromatin remodeling factors can influence DNA repair dynamics.

Personalizing gene therapy in gastric cancer.

Gene therapy was proposed many decades ago as a more straightforward and definitive way of curing human diseases, but only recently technical advancements and improved knowledge have allowed its active development as a broad and promising research field. After the first successes in the cure of genetic and infectious diseases, it has been actively investigated as a means to decrease the burden and suffering generated by cancer. The field of gastric cancer is witnessing an impressive flourishing of studies testing the possibilities and actual efficacy of the many different strategies employed in gene therapy, and overall results seem to be two-sided: while original ideas and innovative protocols are providing extremely interesting contributions with great potential, more advanced-phase studies concluded so far have fallen short of expectations regarding efficacy, although invariably demonstrating little or no toxicity. An overview of the major efforts in this field is provided here, and a critical discussion is presented on the single strategies undertaken and on the overall balance between potentiality and pitfalls.

Gene therapy for lung cancer: practice and promise.

Gene therapy has emerged as an exciting and promising strategy of cancer therapy. Improved molecular biology techniques and a greater understanding of the mechanisms involved in lung cancer pathogenesis allowed a variety of genes to be validated as molecular targets for gene therapy. A variety of gene therapy strategy have been explored in the pre-clinical research. These include replacement of defective tumor suppressor genes, inactivating oncogenes, introducing suicide genes, immunogenic therapy, and antiangiogenesis-based approach. Clinical trials of gene therapy for lung cancer showed the feasibility of delivering a variety of agent as well as highlighted problems with the delivery of therapeutic constructs. Although some may consider the initial results of these novel therapies to be disappointing, they underscore the complexity of these approaches and the likelihood that these approaches will be effective only when used in a coordinated fashion in the proper clinical context. This review provides an update on our current understanding of lung cancer biology and examines several important issues in cancer gene therapy. In addition, recent results of clinical trials of gene therapy for lung cancer are presented.

pRb2/p130 and p107 control cell growth by multiple strategies and in association with different compartments within the nucleus.

It has been recently reported that retinoblastoma family proteins suppress cell growth by regulating not only E2F-dependent mRNA transcription but also rRNA and tRNA transcription and, through HDAC1 recruitment, chromatin packaging. In the present study we report data showing that these various control strategies are correlated, at least in part, with nuclear compartmentalization of retinoblastoma proteins. In a first series of experiments, we showed that pRb2/p130 and p107 are not evenly distributed within the nucleus and that cell cycle-dependent binding with E2F4 changes also as a function of their subnuclear localization. Namely, in the nucleoplasm pRb2/p130-E2F4 complexes are more numerous during G0/G1 while in the nucleolus they increase in S phase. Partially different functions for p107 are suggested since p107-E2F4 complexes in the nucleoplasm are more numerous is S phase with respect to G0/G1 and no cell cycle change is observed in the nucleolus. In a second series of experiments we showed that pRb2/p130, p107, E2F4, and pRb2/p130-HDAC1 complexes are all inner nuclear matrix-associated proteins and localize to sites different from pRb/p105 ones. We provide further evidence of multiple and partially distinct retinoblastoma protein family functional roles during cell cycle. Moreover, our data support emerging evidence for functional interrelationships between nuclear structure and gene expression.

Mandibular metastatic hepatocellular carcinoma: report of a case involving severe and uncontrollable hemorrhage.

Hepatocellular carcinoma (HCC) is a malignant tumor with a marked tendency to spread through the portal system. Metastases from HCC usually involve lungs, surrenal glands, the skeletal and gastroenteric systems, spleen, heart and kidneys. Secondary localizations to the mandible are rare. Generally, bone metastases from HCC appear as osteolytic lesions more likely localized to the ribs, spine, femor, omer, sternum, and then to the mandible. Mandibular metastatic HCC is hemorrhagic in nature because of its hypervascularity. Any diagnostic maneuver that could end in bleeding should be avoided. Non-invasive diagnostic procedures such as computer tomography (CT) scan should be preferred. Among the invasive diagnostic procedures, only fine needle biopsy should be attempted and palliative radiotherapy could be useful for the control of local symptoms. A case report of a hemorrhagic mandibular metastatic HCC that had to be treated surgically, in order to control the severe and profuse bleeding, is presented.

Application of the primer in situ DNA synthesis (PRINS) technique to titer recombinant virus and evaluation of the efficiency of viral transduction.

Titration is an important and critical step in dosing recombinant virus for gene therapy. We present a relatively fast, convenient, and sensitive method that allows for precise quantification of recombinant retrovirus. The method is based on PCR amplification of a foreign gene by the PRINS (primer in situ DNA synthesis) technique. The PRINS technique is based on the sequence-specific annealing of unlabeled oligonucleotide DNA in situ. This oligonucleotide operates as a primer for in situ chain elongation catalyzed by the Taq I polymerase. Using digoxygenin-labeled nucleotides as a substrate for chain elongation, the neo-synthetic DNA is labeled by an FITC-conjugated anti-digoxygenin antibody. To avoid the possibility of false positives, we amplified the puromycin-resistance gene, which is associated with the transgene in the same viral vector and is not normally present in mammalian cells. The retroviral titer was evaluated by counting fluorescein isothiocyanate-positive cells after PRINS labeling, while knowing the number of plated cells that were transduced with different amounts of viral supernatant. A comparable viral concentration of 1 x 10(7) infectious units/mL was found among the retroviruses.

pRb2/p130 gene overexpression induces astrocyte differentiation.

There are many data on the activity of the RB gene in neural differentiation and apoptosis, but the role of pRb2/p130 in neuronal and glial maturation has been far less investigated. To elucidate the role of pRb2/p130 in astrocyte development we overexpressed this protein in astrocytoma and normal astrocyte cultures by adenoviral-mediated gene transfer. In astrocytoma cells, p130/RB2 overexpression resulted in a significant reduction of cell growth and in an increased G(0)/G(1) cell population. We did not observe any induction of programmed cell death as determined by TUNEL reaction. Interestingly, pRb2/p130 overexpression induced astrocyte differentiation. Astrocyte cell cycle arrest and differentiation seemed to proceed through a way distinct from the p53 pathway.

RB2/p130 gene-enhanced expression down-regulates vascular endothelial growth factor expression and inhibits angiogenesis in vivo.

Angiogenesis is an essential step in the progression of tumor formation and development. The switch to an angiogenetic phenotype can occur as a distinct step before progression to a neoplastic phenotype and is linked to genetic changes such as mutations in key cell cycle regulatory genes. The pathogenesis of the angiogenetic phenotype may involve the inactivation of tumor suppressor genes such as the "guardian of the genome," p53, and the cyclin-dependent kinase inhibitor p16. Retinoblastoma family member RB2/p130 encodes a cell cycle regulatory protein and has been found mutated in different tumor types. Overexpression of RB2/p130 not only suppresses tumor formation in nude mice but also causes regression of established tumor grafts, suggesting that RB2/p130 may modulate the angiogenetic balance. We found that induction of RB2/p130 expression using a tetracycline-regulated gene expression system as well as retroviral and adenoviral-mediated gene delivery inhibited angiogenesis in vivo. This correlated with pRb2/p130-mediated down-regulation of vascular endothelial growth factor protein expression both in vitro and in vivo.

A serine 37 mutation associated with two missense mutations at highly conserved regions of p53 affect pro-apoptotic genes expression in a T-lymphoblastoid drug resistant cell line.

The p53 protein accumulates rapidly through post-transcriptional mechanisms following cellular exposure to DNA damaging agents and is also activated as a transcription factor leading to growth arrest or apoptosis. Phosphorylation of p53 occurs after DNA damage thereby modulating its activity and impeding the interaction of p53 with its negative regulator oncogene Mdm2. The serines 15 and 37 present in the amino terminal region of p53 are phosphorylated by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. In order to verify if specific p53 mutations occur in the multi-drug resistance phenotype, we analysed the p53 gene in two T-lymphoblastoid cell lines, CCRF-CEM and its multi-drug-resistant clone CCRF-CEM VLB100, selected for resistance to vinblastine sulfate and cross-resistant to other cytotoxic drugs. Both cell lines showed two heterozygous mutations in the DNA binding domain at codons 175 and 248. The multi-drug resistant cell line, CCRF-CEM VLB100, showed an additional mutation that involves the serine 37 whose phosphorylation is important to modulate the protein activity in response to DNA damage. The effects of these mutations on p53 transactivation capacity were evaluated. The activity of p53 on pro-apoptotic genes expression in response to DNA damage induced by (-irradiation, was affected in the vinblastine (VLB) resistant cell line but not in CCRF-CEM sensitive cell line resulting in a much reduced apoptotic cell death of the multi-drug resistant cells.

BCR-ABL prevents c-jun-mediated and proteasome-dependent FUS (TLS) proteolysis through a protein kinase CbetaII-dependent pathway.

The DNA binding activity of FUS (also known as TLS), a nuclear pro-oncogene involved in multiple translocations, is regulated by BCR-ABL in a protein kinase CbetaII (PKCbetaII)-dependent manner. We show here that in normal myeloid progenitor cells FUS, although not visibly ubiquitinated, undergoes proteasome-dependent degradation, whereas in BCR-ABL-expressing cells, degradation is suppressed by PKCbetaII phosphorylation. Replacement of serine 256 with the phosphomimetic aspartic acid prevents proteasome-dependent proteolysis of FUS, while the serine-256-to-alanine FUS mutant is unstable and susceptible to degradation. Ectopic expression of the phosphomimetic S256D FUS mutant in granulocyte colony-stimulating factor-treated 32Dcl3 cells induces massive apoptosis and inhibits the differentiation of the cells escaping cell death, while the degradation-prone S256A mutant has no effect on either survival or differentiation. FUS proteolysis is induced by c-Jun, is suppressed by BCR-ABL or Jun kinase 1, and does not depend on c-Jun transactivation potential, ubiquitination, or its interaction with Jun kinase 1. In addition, c-Jun-induced FUS proteasome-dependent degradation is enhanced by heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and depends on the formation of a FUS-Jun-hnRNP A1-containing complex and on lack of PKCbetaII phosphorylation at serine 256 but not on FUS ubiquitination. Thus, novel mechanisms appear to be involved in the degradation of FUS in normal myeloid cells; moreover, the ability of the BCR-ABL oncoprotein to suppress FUS degradation by the induction of posttranslational modifications might contribute to the phenotype of BCR-ABL-expressing hematopoietic cells.

Inducible pRb2/p130 expression and growth-suppressive mechanisms: evidence of a pRb2/p130, p27Kip1, and cyclin E negative feedback regulatory loop.

The retinoblastoma family of proteins, pRb/p105, p107, and pRb2/ p130, cooperate to regulate cell cycle progression through the G1 phase of the cell cycle. Each of the family members realize their common goal of G1-S checkpoint regulation through overlapping and unique growth regulatory pathways. We took advantage of a tetracycline-regulated gene expression system to control the expression of RB2/p130 in JC virus-induced hamster brain tumor cells to study in vivo the molecular mechanisms used by pRb2/p130 to elicit its growth-suppressive function. We have previously used this system to demonstrate that induction of pRb/ p130 expression suppresses tumor growth in vivo by overcoming neoplastic transformation mediated by the large T-antigen oncoprotein of JCV (JCV TAg). Here we found that induction of pRb2/p130 in vivo specifically inhibits cyclin A- and cyclin E-associated kinase activity and by doing so induces p27Kip1 levels presumably by inhibiting p27Kip1-targeted proteolysis by cyclin E-Cdk2 phosphorylation of p27Kip1. RB2/p130 induction also decreased cyclin A and the transcription factor E2F-1 while increasing cyclin E at both the transcriptional and protein levels of expression. The growth inhibitory activity of pRb2/p130 also correlated with its E2F-binding capacity. Furthermore, p27Kip1 and pRb2/p130 were found to be targets of the JCV TAg oncoprotein and to interact in vivo with each other independently from the presence of TAg. Interestingly, pRb2/p130 expression negatively modulated the binding of p27Kip1 to JCV TAg. These data suggest that pRb2/p130 and p27Kip1 may cooperate in regulating cellular proliferation, and both may be involved in a negative feedback regulatory loop with cyclin E.

Medroxyprogesterone acetate increases anthracyclines uptake in chronic lymphatic leukemia cells: role of nitric oxide and lipid peroxidation.

Anthracyclines are one of the most used drugs in the therapy of several malignant tumors. Unfortunately, its use is still limited by their cardio-toxicity and by the presence of cancer cells resistant to these drugs. In the present study we evaluated the ability of a chemo-sensitizer agent, MPA (Medroxyprogesterone Acetate), to modify anthracyclines intranuclear uptake in normal leukocytes (NL) and in chronic lymphatic leukemia leukocytes (CLL). Moreover we evaluated the role of lipid peroxidation and nitric oxide (NO) production on antracyclines activity and on their combination with MPA. Our data show that MPA significantly increases anthracyclines uptake only in CLL cells and decreases anthracyclines induced lipid peroxidation.

The RB2/p130 gene: the latest weapon in the war against lung cancer?

Lung cancer is the second cause of death after cardiovascular diseases and is the major cause of cancer deaths in the Western world. Large scale screening trials conducted 15-20 years ago using chest X-rays and sputum cytology were able to detect stage I cancers but failed to impact on survival. This is because of the early metastatic potential of small primary tumors. It is important then to detect lung cancer at an earlier stage, studying and identifying genetic lesions that could indicate a new target(s) for gene therapy. The retinoblastoma-related gene pRb2/p130, a new tumor suppressor gene cloned in 1993, is emerging as one of the candidate markers and targets for gene therapeutic approach. Effective genetic therapy requires both a genetic material to be used therapeutically and a means to deliver it. A scope for this review is to examine some of the gene delivery systems mostly used, discussing their weaknesses and strengths, and to discuss the role of pRb2/p130 in lung cancer.

Genetic alterations of the retinoblastoma-related gene RB2/p130 identify different pathogenetic mechanisms in and among Burkitt's lymphoma subtypes.

Alterations of cell cycle-associated genes probably contribute to the pathogenesis of Burkitt's Lymphoma (BL), in addition to c-myc translocation. Mutations disrupting the nuclear localization signal of the retinoblastoma-related gene RB2/p130 have been documented recently in BL cell lines and primary tumors. Given the importance of the RB2/p130 gene in controlling cell growth, mutations of this gene may result in uncontrolled cell proliferation. We tested the expression and genomic organization of the RB2/p130 gene in relation to the proliferative features of a series of BL samples collected from the endemic and sporadic regions, regardless of whether the samples were acquired immune deficiency syndrome (AIDS)-related. The expression of the Rb2/p130, p107, and cell proliferation-related proteins (cyclin A and B) was determined by immunohistochemistry. The structures of exons 19 through 22 of the RB2/p130 gene, encoding for the B domain and C terminus, were analyzed by polymerase chain reaction (PCR) analysis and single-strand conformation polymorphism (SSCP) technique. The direct PCR products were sequenced to identify the actual mutations. Our results suggest that BL is composed of a mixture of molecular types with distinct genetic and phenotypic patterns, probably resulting from different pathogenetic mechanisms. In endemic BL, the RB2/p130 gene is mutated in most of the cases, and the protein is restricted to the cytoplasm. In AIDS-related BL, high levels of nuclear expression of the wild-type pRb2/p130, p107, and cell proliferation-related proteins were detected. This finding is in line with the molecular mechanisms observed in virus-linked oncogenesis. Sporadic BLs were mainly characterized by the low nuclear values of the wild-type pRb2/p130 and, conversely, the high values of p107. The increased cell proliferation due to different alterations of cell growth control by Rb-related proteins may be the first step in lymphomagenesis, during which additional genetic changes, including missense mutations of c-myc, may subsequently occur.

Mutations in the retinoblastoma-related gene RB2/p130 in lung tumors and suppression of tumor growth in vivo by retrovirus-mediated gene transfer.

The retinoblastoma (Rb) family consists of the tumor suppressor pRb/p105 and related proteins p107 and pRb2/p130. Recent immunohistochemical studies of the retinoblastoma family of proteins in 235 specimens of lung cancer show the tightest inverse association between the histological grading in the most aggressive tumor types and pRb2/p130. This led us to study a panel of human lung cancers for mutations in the RB2/p130 gene. Mutations in the Rb-related gene RB2/p130 were detected in 11 of 14 (78.5%) primary lung tumors by single-strand conformation polymorphism and sequence analysis. A Moloney leukemia virus-based retroviral system was set up, and a comparable viral concentration of 1 x 10(7) infectious units/ml was obtained. Retrovirus-mediated delivery of wild-type RB2/p130 to the lung tumor cell line H23 potently inhibited tumorigenesis in vitro and in vivo, as shown by the dramatic growth arrest observed in a colony assay and the suppression of anchorage-independent growth potential and tumor formation in nude mice. The tumors transduced with the RB2/p130 retrovirus diminished in size after a single injection, and a 12-fold reduction in tumor growth after RB2/p130 transduction compared with the Pac-transduced tumors (92% reduction, P = 0.003) and lacZ-transduced tumors (93% reduction, P < 0.001) was found to be statistically significant. These findings provide the missing confirmation that RB2/p130 is a "bona fide" tumor suppressor gene and strengthen the hypothesis that it may be a candidate for cancer gene therapy for lung cancer.

Genetic alterations disrupting the nuclear localization of the retinoblastoma-related gene RB2/p130 in human tumor cell lines and primary tumors.

The prototypic tumor suppressor gene, the retinoblastoma gene (RB/ p105), is mutated in a variety of human tumors. However, to date, mutational data on retinoblastoma family members p107 and RB2/p130 in tumors is lacking. We studied the expression of pRb2/p130 by immunocytochemistry and Western blot analysis in a panel of human osteosarcoma and lymphoid cell lines. Only the lymphoid cell lines showed an abnormal cytoplasmic localization of pRb2/p130, suggesting possible alterations within the region of nuclear localization signaling. We screened these cell lines for genetic alterations of the RB2/p130 gene in the region of the putative bipartite nuclear localization signal (NLS). This region is highly homologous with that of the RB/p105 gene. In addition, we screened four primary Burkitt's lymphomas for genetic alterations in the RB2/p130 gene. Naturally occurring mutations, which disrupt the putative bipartite NLS, were found in lymphoma cell lines and primary tumors, but not in the osteosarcoma cell lines, where normal nuclear localization of the protein was detectable. Site-directed mutagenesis and transfection assay using NLS mutants displayed markedly reduced biological activity as measured by flow cytometric analysis. This study clearly describes RB2/ p130 as an important target for mutations and subsequent inactivation in lymphoma pathogenesis, thus validating that RB2/p130 is a classical tumor suppressor gene.