Candace M. Howard

A UNIQUE DOMAIN OF PRB2/P130 ACTS AS AN INHIBITOR OF CDK2 KINASE ACTIVITY

The Cdk2 kinase has long been known to be involved in the progression of mammalian cells past the G1 phase restriction point and through DNA replication in the cell cycle. The Rb family of proteins, consisting of pRb, p107, and pRb2/p130, has also been shown to monitor progression of G1 phase, mostly through their interaction with E2F family members. p107 is able to inhibit Cdk2 kinase activity through this interaction via a p21-related domain present in the C terminus of the protein. We show here that pRb2/p130 also possesses this activity, but through a separate domain. Moreover, we correlate the increased expression of pRb2/p130 during various cellular processes with the decreased kinase activity of Cdk2. We hypothesize that pRb2/p130 may act not only to bind and modify E2F activity, but also to inhibit Cdk2 kinase activity in concert with p21 in a manner different from p107.

Adenoviral RB2/p130 Gene Transfer Inhibits Smooth Muscle Cell Proliferation and Prevents Restenosis After Angioplasty

Smooth muscle cell (SMC) proliferation that results in neointima formation is implicated in the pathogenesis of atherosclerotic plaques and accounts for the high rates of restenosis that occur after percutaneous transluminal coronary angioplasty, a widespread treatment for coronary artery disease. Endothelial lesions trigger intense proliferative signals to the SMCs of the subintima, stimulating their reentry into the cell cycle from a resting G(0) state, resulting in neointima formation and vascular occlusion. Cellular proliferation is negatively controlled by growth-regulatory or tumor-suppressor genes, or both, such as the retinoblastoma gene family members (RB/p105, p107, RB2/p130). In the present study, we show that RB2/p130 inhibited SMC proliferation in vitro and in vivo. We used the rat carotid artery model of restenosis to demonstrate that adenovirus-mediated localized arterial transduction of RB2/p130 at the time of angioplasty significantly reduced neointimal hyperplasia and prevented restenosis. Furthermore, the ability of pRb2/p130 to block proliferation correlated with its ability to bind and sequester the E2F family of transcription factors, which are important mediators of cell cycle progression. These results imply that RB2/p130 could be an important target for vascular gene therapy.

Ectopic expression of pRb2/p130 suppresses the tumorigenicity of the c-erbB-2-overexpressing SKOV3 tumor cell line

We investigated the in vitro and in vivo effects of the ectopic expression of the pRb2/p130 cell cycle regulator on c-erbB-2-associated tumorigenicity. SKOV3 ovarian cancer cells, which display c-erbB-2 gene amplification and oncoprotein (p185HER2) overexpression, were stably transfected with a plasmid containing the coding sequence for human wild-type pRb2/p130 (wtRb2), or with pcDNA3 empty vector. Three wtRb2-transfected clones (cl. 24, cl. 49, cl. 100) and one empty vector-transfected clone (cl. mock) were randomly picked and further analysed. Western blot analysis revealed high levels of pRb2/p130 in the three clones compared to mock cells. Levels of p185HER2 and the extent of its tyrosine phosphorylation were similar in all transfectant clones, as were levels of pRb1 and p107. In anchorage-independent growth assays, the number of colonies from wtRb2 clone-transfectants was about 90% less than that arising from mock cells (P<0.001). Tumor take rates of the three wtRb2-transfected clones xenografted in nu/nu mice were much lower than those of mock cells, and tumor volume was decreased by 80% (P<0.001). A mutant version of pRb2/p130 deleted of the pocket region (mut-Rb2) was also transfected into SKOV3 cells and studied in parallel with the wtRb2-transfected and pcDNA empty vector-transfected bulk populations. mut-Rb2 transfected cells showed no inhibition of in vitro colony formation and were fully tumorigenic. Together, these findings indicate that Rb2 acts as a tumor suppressor gene in vivo and in vitro in SKOV3 cells and that the intact pocket region is required for the suppressor activity.

Expression of Cell Cycle–Regulated Proteins pRB2/p130, p107, E2F4, p27, and pCNA in Salivary Gland Tumors: Prognostic and Diagnostic Implications

The retinoblastoma family consists of the tumor suppressor nuclear phosphoprotein pRb/p105 and related proteins p107 and pRb2/p130. Recent immunohistochemical studies of the retinoblastoma family of proteins in lung and endometrial cancer and choroidal melanomas show a tight inverse correlation between the histologic grading in the most aggressive tumor types and pRb2/p130 expression. This led us to investigate the role of pRb2/p130 in salivary tumors. We studied the expression of pRb2/p130, p107, E2F4, p27, and PcNA by immunohistochemistry in a panel of 44 salivary gland tumors. We found a direct correlation between the cytoplasmic expression of pRb2/p130 and tumor grading and the presence of metastasis that was highly statistically significant (P < 0.001). Additionally, increased cytoplasmic pRb2/p130 expression was significantly correlated with a decreased probability of survival (P < 0.001). Interestingly, p107 nuclear expression showed a strong direct correlation when compared with the same variables. pRb2/p130 showed the highest percentage of undetectable nuclear levels in the specimens examined and the tightest inverse correlation (P < 0.0001) with both the histologic grading and pCNA expression in malignant salivary tumors. Additionally, E2F4 showed an identical localization pattern as to that of pRb2/p130. These data suggests an important role for pRb2/p130 in the pathogenesis and progression of certain salivary gland cancers.

Maxillary constriction treated by a new palatal distractor device: surgical and occlusal evaluations of 10 patients.

Purpose:To study the changes in maxillary dimensions after the application of a new distractor on 10 adult patients affected by severe palatal constriction. Materials and Methods:The palatal distractor device was made of a Rematitan titanium expansion screw (Dentaurum) welded to 2 titanium miniplates (LEIBINGER) on patients models. The device was applied using four 8-mm screws, activated 0.20 mm 4 times a day, and blocked for 4 months. The intermolar, interpremolar, and intercanine distances were measured before the palatal distractor device application and 1 week and 4 months after activation. Changes in dental angulation in the frontal plane, the intermolar, and the interpremolar angle variations were measured. Results:There was an increase of the arch perimeter, well correlated with the expansion at dental level, resulting in teeth crowding resolution. The changes in dental angulation in the frontal plane were minimal, indicating mainly a skeletal movement. Conclusions:The device produces mainly a skeletal movement and a minimal dental angulation movement.

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.

Eating Green: Shining Light on the Use of Dietary Phytochemicals as a Modern Approach in the Prevention and Treatment of Head and Neck Cancers

Enthusiasm for the use of dietary bioactive compounds as chemopreventive agents and adjuvants for current therapies has increased laboratory research conducted on several types of cancers including Head and Neck Squamous Cell Carcinoma (HNSCC). The green chemoprevention movement is a modern approach to highlight healthy lifestyle changes that aim to decrease the incidence of HNSCC. A healthy diet can be an effective way to prevent the development of oral cancers. Discovery of the naturally occurring plant based compounds called phytochemicals has facilitated the development of new treatment strategies for patients that are at risk for, or have developed HNSCC. Many of these compounds have been shown to elicit very potent anti-carcinogenic properties. While there are many compounds that have been studied, the compounds from two specific categories of phytochemicals, phenolics (resveratrol, EGCG, curcumin, quercetin, and honokiol) and glucosinolates (sulforaphane, PEITC and BITC), are emerging as potent and effective inhibitors of oral carcinogenesis. These compounds have been shown to inhibit HNSCC growth through a variety of mechanisms. Research has demonstrated that these compounds can regulate cancer cell proliferation through the regulation of multiple cell signaling pathways. They can impede cell cycle progression, induce differentiation and apoptosis, prevent angiogenesis, and inhibit cancer cell invasive and metastatic properties. They can protect normal cells during treatment and reduce the damage caused by chemotherapy and radiotherapy. This review aims to provide an overview of some of the most effective phytochemicals that have the potential to successfully prevent and treat head and neck squamous cell carcinoma.

Cutting Edge Therapies for Cancer in the 21st Century

Cancer is a broad group of diseases involving unregulated cell growth with elevated death rates since more people live in old age with mass lifestyle changes occurring in the developing world. The causes of cancer are diverse, complex and still only partially understood. The chances of surviving the disease vary remarkably by the type and location of the malignancy and the extent of disease at the start of treatment. Early cancer detection is proviing to be a valid approach. Cancer can be detected in a number of ways, including the presence of certain signs and symptoms, screening tests or medical imaging. Cancer therapy is dynamically changing and revision and change in patient management is constant. Cancer is routinely treated with chemotherapy, radiation therapy and surgery. Tailored cancer targeted therapy is becoming an emerging objective of today.

Using a Commercial Ultrasound Contrast Agent for Viral‐Mediated Gene Transfer In Vitro and In Vivo

This study evaluated the feasibility of site‐specific gene delivery mediated by diagnostic ultrasound using genes encapsulated in commercially available ultrasound contrast agents in vitro and in vivo. Five different commercially available contrast agents were tested in vitro for their ability to enclose an adenoviral vector carrying GFP. Prostate cancer cells (DU 145) or non small cell lung cancer cells (H23) were plated in 80 culture wells and insonified at 207 or 535 kPa peak negative pressure for 1 min after administration of 0.1 ml of bubbles reconstituted with the viral vector. Experiments were repeated with the delivery vehicle incubated with complement to inactivate unenclosed Adeno‐GFP and with controls. After 24 hours transduction efficiency was demonstrated by fluorescent microscopy. In vivo 15 nude mice with 21 melanoma tumors (DB‐1) implanted received 0.1 ml injections of contrast. Mice were split into 3 control and 4 active groups and ultrasound was performed for 4 min at 4 MHz using an Apli...

Platelet-Rich Fibrin (PRF) in Implants Dentistry in Combination with New Bone Regenerative Flapless Technique: Evolution of the Technique and Final Results.

Abstract Most common techniques for alveolar bone augmentation are guided bone regeneration (GBR) and autologous bone grafting. GBR studies demonstrated long-term reabsorption using heterologous bone graft. A general consensus has been achieved in implant surgery for a minimal amount of 2 mm of healthy bone around the implant. A current height loss of about 3-4 mm will result in proper deeper implant insertion when alveolar bone expansion is not planned because of the dome shape of the alveolar crest. To manage this situation a split crest technique has been proposed for alveolar bone expansion and the implants’ insertion in one stage surgery. Platelet-rich fibrin (PRF) is a healing biomaterial with a great potential for bone and soft tissue regeneration without inflammatory reactions, and may be used alone or in combination with bone grafts, promoting hemostasis, bone growth, and maturation. Aim The aim of this study was to demonstrate the clinical effectiveness of PRF combined with a new split crest flapless modified technique in 5 patients vs. 5 control patients. Materials and methods Ten patients with horizontal alveolar crests deficiency were treated in this study, divided into 2 groups: Group 1 (test) of 5 patients treated by the flapless split crest new procedure; Group 2 (control) of 5 patients treated by traditional technique with deeper insertion of smaller implants without split crest. The follow-up was performed with x-ray orthopantomography and intraoral radiographs at T0 (before surgery), T1 (operation time), T2 (3 months) and T3 (6 months) post-operation. Results All cases were successful; there were no problems at surgery and post-operative times. All implants succeeded osteointegration and all patients underwent uneventful prosthetic rehabilitation. Mean height bone loss was 1 mm, measured as bone-implant most coronal contact (Δ-BIC), and occurred at immediate T2 post-operative time (3 months). No alveolar bone height loss was detected at implant insertion time, which was instead identified in the control group because of deeper implant insertion. Conclusion This modified split crest technique combined with PRF appears to be reliable, safe, and to improve the clinical outcome of patients with horizontal alveolar crests deficiency compared to traditional implanting techniques by avoiding alveolar height-loss related to deeper insertion of smaller implants.