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.

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.

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.

Stretch-mediated release of angiotensin II induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell.

Physical forces activate apoptosis and gene expression, but the mechanism is unknown. For this purpose, adult myocytes were stretched in an equibiaxial stretch apparatus and the magnitude of cell death was examined 4 and 24 h later. The possibility of stretch-mediated activation of p53 and p53-dependent genes was evaluated at 30 min, 2, 4, 8, and 24 h. Myocyte apoptosis increased by 4.4- and 7.6-fold at 4 and 24 h after stretch. p53 binding to the promoter of angiotensinogen, AT1 receptor, and Bax also increased. Expression of angiotensinogen, AT1 receptor, p53, and Bax increased and Bcl-2 decreased in stretched myocytes. The changes in AT1 receptor, p53, Bax, and Bcl-2 became more apparent with the duration of stretch. Angiotensin II concentration in the medium increased at 10 min, reaching maximal levels at 1 and 20 h. The AT1 blocker, losartan, abolished apoptosis in stretched myocytes. Myocyte volume was not influenced by stretch. In conclusion, stretch-mediated release of angiotensin II is coupled with apoptosis and the activation of p53 which may be responsible for the prolonged upregulation of the local renin-angiotensin system and the increased susceptibility of myocytes to undergo apoptosis.

Activated Raf-1 causes growth arrest in human small cell lung cancer cells.

Small cell lung cancer (SCLC) accounts for 25% of all lung cancers, and is almost uniformly fatal. Unlike other lung cancers, ras mutations have not been reported in SCLC, suggesting that activation of ras-associated signal transduction pathways such as the raf-MEK mitogen-activated protein kinases (MAPK) are associated with biological consequences that are unique from other cancers. The biological effects of raf activation in small cell lung cancer cells was determined by transfecting NCI-H209 or NCI-H510 SCLC cells with a gene encoding a fusion protein consisting of an oncogenic form of human Raf-1 and the hormone binding domain of the estrogen receptor (DeltaRaf-1:ER), which can be activated with estradiol. DeltaRaf-1:ER activation resulted in phosphorylation of MAPK. Activation of this pathway caused a dramatic loss of soft agar cloning ability, suppression of growth capacity, associated with cell accumulation in G1 and G2, and S phase depletion. Raf activation in these SCLC cells was accompanied by a marked induction of the cyclin-dependent kinase (cdk) inhibitor p27(kip1), and a decrease in cdk2 protein kinase activities. Each of these events can be inhibited by pretreatment with the MEK inhibitor PD098059. These data demonstrate that MAPK activation by DeltaRaf-1:ER can activate growth inhibitory pathways leading to cell cycle arrest. These data suggest that raf/MEK/ MAPK pathway activation, rather than inhibition, may be a therapeutic target in SCLC and other neuroendocrine tumors.

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.

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.

Retinoblastoma-related protein pRb2/p130 and suppression of tumor growth in vivo.

BACKGROUND: The RB/p105 and p107 genes of the retinoblastoma family are tumor suppressor genes whose proteins are inactivated by interaction with T-antigen proteins encoded by polyomaviruses (e.g., simian virus 40 and human JC virus), which have been found to be highly tumorigenic in animals. A variety of indirect evidence suggests that another member of the retinoblastoma gene family, RB2/p130, is also a tumor suppressor gene. To investigate the putative tumor suppressor activity of RB2/p130 more directly, we utilized a tetracycline-regulated gene expression system to control expression of the encoded protein pRb2/p130 in JC virus-induced hamster brain tumor cells and to study the effects of pRb2/p130 on the growth of such tumor cells in nude mice. The ability of pRb2/p130 to interact with JC virus T antigen was also studied. METHODS: Northern blot hybridization analyses were performed on samples of total cellular RNA to measure RB2/p130 and beta-actin messenger RNA levels. Immunoprecipitation and western blot analyses were used to determine T-antigen and pRb2/p130 protein levels and to assess the phosphorylation status of these proteins. Tumor cells were injected subcutaneously into nude mice, and tumor growth, with or without induced expression of pRb2/p130, was monitored. RESULTS: Induction of pRb2/p130 expression brought about a 3.2-fold, or 69% (95% confidence interval = 64%-73%), reduction in final tumor mass in nude mice. We also demonstrated that JC virus T antigen binds hypophosphorylated pRb2/p130 and that stimulation of pRb2/p130 expression overcomes cellular transformation mediated by this antigen. CONCLUSION: Our findings support the hypothesis that RB2/p130 is a tumor suppressor gene.

Bdp, a new member of a family of DNA-binding proteins, associates with the retinoblastoma gene product.

We have cloned a gene, BDP, encoding a protein with homology to the retinoblastoma-binding proteins Rbp1 and Rbp2. It also has homology to DNA-binding proteins such as Bright, a B-cell-specific trans-activator, and the Drosophila melanogaster dead ringer gene product. Like MyoD, Bdp binds to the COOH-terminal region of pRb through its conserved region and to hypophosphorylated pRb. It also binds to the MAR of the immunoglobulin heavy-chain locus. Thus Bdp may contribute to the transcriptional regulation of genes involved in differentiation and tissue-specific expression.

The cdc-2-related kinase, PISSLRE, is essential for cell growth and acts in G2 phase of the cell cycle.

Mammalian cell cycle progression is regulated by several protein kinases that are activated by cyclically expressed proteins called cyclins. These cyclin-dependent kinases, the prototype of which is the cdc2 mitosis-promoting kinase, are known to phosphorylate substrates the modified status of which is critical for the cell to progress into sequential phases of the cycle. Recently, a new cdc2-related protein kinase has been discovered. PISSLRE, named with respect to its homology to the cdc2 PSTAIRE amino acid domain. Here we report that by using both antisense and dominant-negative mutant constructs of PISSLRE when overexpressed in U2OS cells, a growth suppression is found. Furthermore, the dominant negative forms of PISSLRE halt cell cycle progression in G2-M. Therefore, PISSLRE is essential for cellular proliferation, and its effect is exerted in G2-M. This describes the first evidence since cdc2 of a cdc2-related kinase acting through G2-M.

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.

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.

Mutations in the retinoblastoma-related gene RB2/p130 in primary nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an endemic cancer in southern China and northern Africa, and its pathogenesis is not yet well defined at the molecular level. Although the involvement of p53 and of the retinoblastoma gene (RB/p105) in NPC has been well studied, there is paucity of mutational data regarding the retinoblastoma-related gene RB2/p130 in primary tumors and particularly in NPC. We have shown previously that RB2/p130 could be rearranged in a nasopharyngeal cell line. In the present study, we screened by single-strand conformation polymorphism and sequence analysis the retinoblastoma-related gene RB2/p130 for mutations within exons 19-22. Mutations in the RB2/p130 gene were detected in 3 of 10 primary human NPCs from Northern Africa (30%). These findings, along with previous data showing that genetic replacement of RB2/p130 restores a normal growth pathway in the nasopharyngeal cell line Hone-1, strengthen the hypothesis that genetic changes of RB2/p130 may be involved in the development and/or progression of nasopharyngeal cancer and suggest that RB2/p130 could be considered a tumor suppressor gene and may be a candidate for novel gene therapeutic approaches for NPC.

p130/pRb2 has growth suppressive properties similar to yet distinctive from those of retinoblastoma family members pRb and p107.

The retinoblastoma tumor suppressor gene product, as well as its related protein p107, has been shown clearly to exert its growth suppressive effects in a cell cycle dependent manner. In this study we demonstrate that the introduction of our recently cloned Rb family member p130/pRb2 causes growth arrest in three tumor cell lines. In addition, in the nasopharyngeal carcinoma derived cell line HONE-1, we identified a low level of expression of p130/pRb2, possibly due to gene rearrangement, and a drastic reduction in proliferation upon introduction of a constitutive active p130/pRb2 complementary DNA clone. Furthermore, we were able to dissect distinct properties of the Rb family by demonstrating that p130/pRb2 inhibits proliferation of the glioblastoma cell line T98G, which is resistant to the growth suppressive effects of both pRb and p107. Our studies demonstrate that the Rb family proteins identified to date may complement each other but they are not fully functionally redundant.

Functional analysis of pRb2/p130 interaction with cyclins.

The retinoblastoma (Rb) family consists of the tumor suppressor pRb and related proteins p107 and pRb2/p130. Ectopic expression of pRb and p107 results in a growth arrest of sensitive cells in the G1 phase of the cell cycle. We demonstrated here that the growth-suppressive properties of pRb2/p130 were also specific for the G1 phase. The A-, E-, and D-type cyclins as well as transcription factor E2F1 and the E1A viral oncoprotein were able to rescue the pRb2/p130-mediated G1 growth arrest in SAOS-2 cells. The rescue with cyclins A and E correlated with their physical interaction with pRb2/p130, which surprisingly has been found to occur over all phases of the cell cycle. The phosphorylation status as well as the kinase activity associated with pRb2/p130 dramatically increased near the G1-S-phase transition. This suggests that, like the other Rb family members, pRb and p107, the phosphorylation of pRb2/p130 is controlled by the cell cycle machinery and that pRb2/p130 may indeed be another key G1-S-phase regulator.

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.

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.

PISSLRE, a human novel CDC2-related protein kinase.

The cell division cycle have been shown to be regulated by a closely-related family of protein kinases named CDKs (by cyclin-dependent kinases). Using a PCR-based cloning technique, we have isolated cDNAs encoding a human CDC2-related protein kinase. The full-length cDNA accommodates an open reading frame that does not contain any ATG initiation codon upstream of the sequence encoding the catalytic domain of this putative kinase. Three putative non-ATG initiation codons have been detected. Starting at the most 5' non-ATG initiation site, the encoded product is 316 amino acids long with a predicted molecular weight of 35.8 kDa. Analysis of the deduced amino acid sequence showed it to contain the XI subdomains present in all known protein kinases and a PSTAIRE-like motive, PISSLRE, which temporarily names this kinase. PISSLRE is most related to p58/GTA (55% identity in the catalytic domain), the galactosyl transferase associated protein, which has been shown to inhibit entry into S-phase when over-expressed in CHO cells. PISSLRE shares 38-45% identity with all CDKs and contains the regulatory Tyr and Thr residues present in most of the members of the CDK family of protein kinases, which suggests similar modes of regulation. PISSLRE is expressed in all human tissues tested, including those which contain high proportion of terminally differentiated cells. However, the levels of the PISSLRE transcripts are dissimilar among different tissues.