Biomedical subjects

R R Weichselbaum

Publications and source records attributed to R R Weichselbaum.

At least 37 records · Page 2Linked to original sources

PubMed2001

cdc2 cyclin-dependent kinase binds and phosphorylates herpes simplex virus 1 U(L)42 DNA synthesis processivity factor.

Earlier studies have shown that cdc2 kinase is activated during herpes simplex virus 1 infection and that its activity is enhanced late in infection even though the levels of cyclin A and B are decreased below levels of detection. Furthermore, activation of cdc2 requires the presence of infected cell protein no. 22 and the U(L)13 protein kinase, the same gene products required for optimal expression of a subset of late genes exemplified by U(S)11, U(L)38, and U(L)41. The possibility that the activation of cdc2 and expression of this subset may be connected emerged from the observation that dominant negative cdc2 specifically blocked the expression of U(S)11 protein in cells infected and expressing dominant negative cdc2. Here we report that in the course of searching for a putative cognate partner for cdc2 that may have replaced cyclins A and B, we noted that the DNA polymerase processivity factor encoded by the U(L)42 gene contains a degenerate cyclin box and has been reported to be structurally related to proliferating cell nuclear antigen, which also binds cdk2. Consistent with this finding, we report that (i) U(L)42 is able to physically interact with cdc2 at both the amino-terminal and carboxyl-terminal domains, (ii) the carboxyl-terminal domain of U(L)42 can be phosphorylated by cdc2, (iii) immunoprecipitates obtained with anti U(L)42 antibody contained a roscovitine-sensitive kinase activity, (iv) kinase activity associated with U(L)42 could be immunodepleted by antibody to cdc2, and (v) U(L)42 transfected into cells associates with a nocodazole-enhanced kinase. We conclude that U(L)42 can associate with cdc2 and that the kinase activity has the characteristic traits of cdc2 kinase.

Amino Acid Sequence↗

PubMed2001

Inhibition of glioma cell growth and tumorigenic potential by CCN3 (NOV).

AIMS: To establish whether the ectopic expression of CCN3 (NOV) in glioma cells can interfere with their tumorigenic potential and assess its potential value in molecular medicine. METHODS: Glioma cell lines were used to assess whether differences in the degree of intracellular communication induced by the expression of the gap junction protein connexin 43 (Cx43) is related to the differential expression of CCN3 (NOV). The antiproliferative activity of rat CCN3 (rCCN3; NOV) in glioma cells, has been assessed both in vitro and in vivo with glioma cell lines expressing different amounts of CCN3 (NOV). RESULTS: Upon ectopic expression of Cx43, the growth of C6 glioma cells is decreased. An increase of CCN3 (NOV) expression matches the reduced tumorigenic potential of these transfected cells. The localisation of CCN3 (NOV) is affected by the increased expression of Cx43 in the Cx-13 transfected cells, in which it is detected at areas of cell-cell contact. In a xenograft model, CCN3 (NOV) transfected glioma cells were found to induce tumours to a lesser degree than their parental counterparts, which do not express detectable amounts of CCN3 (NOV). CONCLUSIONS: Previous observations had suggested an inverse relation between CCN3 (NOV) expression in glioma cells and their tumorigenicity. These results establish a direct association between the establishment of functional gap junctional intercellular communication and the expression of rCCN3 (NOV). In addition to a negative effect on murine and human cell growth, CCN3 (NOV) has antiproliferative activity on tumour cells in vivo. Thus, the antiproliferative activity of the CCN3 (NOV) protein might involve reorganisation of cellular contacts that play a crucial role in tumorigenesis. The antiproliferative activity of CCN3 (NOV) established in this work sets the stage for the potential use of CCN proteins in molecular oncology.

Animals↗

PubMed2001

Treatment of head and neck and esophageal xenografts employing Alimta and concurrent ionizing radiation.

We examined the interaction between Alimta and ionizing radiation (IR) as a potential strategy to enhance the therapeutic ratio of combined-modality cancer treatment. Mice bearing human esophageal adenocarcinoma xenografts (Seg-1) or squamous cell carcinoma xenografts (SQ-20B) were treated with Alimta and IR employing a fractionated treatment schedule. Treatment with Alimta alone slowed the growth of Seg-1 but not SQ-20B tumors compared with control tumors. In Seg-1 xenografts combined treatment with Alimta and IR produced significant tumor growth inhibition compared with Alimta alone or IR alone. In SQ-20B xenografts, treatment with Alimta did not enhance IR-mediated tumor growth inhibition suggesting that sensitivity to Alimta is necessary for an interactive cytotoxic effect with IR. The present data suggest the potential clinical efficacy of combining Alimta administration with radiotherapy for Alimta-sensitive cells and indicate that further testing needs to be conducted to optimize the dosing schedule to enhance the interaction between the therapeutic agents.

Adenocarcinoma↗

PubMed2000

NM-3, an isocoumarin, increases the antitumor effects of radiotherapy without toxicity.

We examined the effects of a new antiangiogenic isocoumarin, NM-3, as a radiation modifier in vitro and in vivo. The present studies demonstrate that NM-3 is cytotoxic to human umbilical vein endothelial cells (HUVECs) but not to Lewis lung carcinoma (LLC) cells nor Seg-1, esophageal adenocarcinoma cells, in clonogenic survival assays. When HUVEC cultures are treated with NM-3 combined with ionizing radiation (IR), additive cytotoxicity is observed. In addition, the combination of NM-3 and IR inhibits HUVEC migration to a greater extent than either treatment alone. The effects of treatment with NM-3 and IR were also evaluated in tumor model systems. C57BL/6 female mice bearing LLC tumors were given injections for 4 consecutive days with NM-3 (25 mg/kg/day) and treated with IR (20 Gy) for 2 consecutive days. Combined treatment with NM-3 and IR significantly reduced mean tumor volume compared with either treatment alone. An increase in local tumor control was also observed in LLC tumors in mice receiving NM-3/IR therapy. When athymic nude mice bearing Seg-1 tumor xenografts were treated with NM-3 (100 mg/kg/day for 4 days) and 20 Gy (four 5 Gy fractions), significant tumor regression was observed after combined treatment (NM-3 and IR) compared with IR alone. Importantly, no increase in systemic or local tissue toxicity was observed after combined treatment (NM-3 and IR) when compared with IR alone. The bioavailability and nontoxic profile of NM-3 suggests that the efficacy of this agent should be tested in clinical radiotherapy.

Adenocarcinoma↗

PubMed2000

Quantitative analysis of DNA array autoradiographs.

DNA arrays and chips are powerful new tools for gene expression profiling. Current arrays contain hundreds or thousands of probes and large scale sequencing and screening projects will likely lead to the creation of global genomic arrays. DNA arrays and chips will be key in understanding how genes respond to specific changes of environment and will also greatly assist in drug discovery and molecular diagnostics. To facilitate widespread realization of the quantitative potential of this approach, we have designed procedures and software which facilitate analysis of autoradiography films with accuracy comparable to phosphorimaging devices. Algorithms designed for analysis of DNA array autoradiographs incorporate 3-D peak fitting of features on films and estimation of local backgrounds. This software has a flexible grid geometry and can be applied to different types of DNA arrays, including custom arrays.

Algorithms↗

PubMed2000

The role of cdc2 in the expression of herpes simplex virus genes.

Earlier reports have shown that cdc2 kinase is activated in cells infected with herpes simplex virus 1 and that the activation is mediated principally by two viral proteins, the infected cell protein 22 (ICP22) and the protein kinase encoded by U(L)13. The same proteins are required for optimal expression of a subset of late (gamma(2)) genes exemplified by U(S)11. In this study, we used a dominant-negative cdc2 protein to determine the role of cdc2 in viral gene expression. We report the following. (i) The cdc2 dominant-negative protein had no effect in the synthesis and accumulation of at least two alpha-regulatory proteins (ICP4 and ICP0), two beta-proteins (ribonucleotide reductase major subunit and single-stranded DNA-binding protein), and two gamma(1)-proteins (glycoprotein D and viral protease). U(S)11, a gamma(2)-protein, accumulated only in cells in which cdc2 dominant-negative protein could not be detected or was made in very small amounts. (ii) The sequence of amino acids predicted to be phosphorylated by cdc2 is present in at least 27 viral proteins inclusive of the regulatory proteins ICP4, ICP0, and ICP22. In in vitro assays, we demonstrated that cdc2 specifically phosphorylated a polypeptide consisting of the second exon of ICP0 but not a polypeptide containing the sequence of the third exon as would be predicted from the sequence analysis. We conclude that cdc2 is required for optimal expression of a subset of gamma(2)-proteins whose expression is also regulated by the viral proteins (ICP22 and U(L)13) that mediate the activation of cdc2 kinase.

CDC2 Protein Kinase↗

PubMed2000

The breast cancer susceptibility gene BRCA1 is required for subnuclear assembly of Rad51 and survival following treatment with the DNA cross-linking agent cisplatin.

Mutations in breast cancer tumor susceptibility genes, BRCA1 and BRCA2, predispose women to early onset breast cancer and other malignancies. The Brca genes are involved in multiple cellular processes in response to DNA damage including checkpoint activation, gene transcription, and DNA repair. Biochemical interaction with the recombinational repair protein Rad51 (Scully, R., Chen, J., Ochs, R. L., Keegan, K., Hoekstra, M., Feunteun, J., and Livingston, D. M. (1997) Cell 90, 425-435), as well as genetic evidence (Moynahan, M. E., Chiu, J. W., Koller, B. H., and Jasin, M. (1999) Mol. Cell 4, 511-518 and Snouwaert, J. N., Gowen, L. C., Latour, A. M., Mohn, A. R., Xiao, A., DiBiase, L., and Koller, B. H. (1999) Oncogene 18, 7900-7907), demonstrates that Brca1 is involved in recombinational repair of DNA double strand breaks. Using isogenic Brca1(+/+) and brca1(-/-) mouse embryonic stem (ES) cell lines, we investigated the role of Brca1 in the cellular response to two different categories of DNA damage: x-ray induced damage and cross-linking damage caused by the chemotherapeutic agent, cisplatinum. Immunoflourescence studies with normal and brca1(-/-) mutant mouse ES cell lines indicate that Brca1 promotes assembly of subnuclear Rad51 foci following both types of DNA damage. These foci are likely to be oligomeric complexes of Rad51 engaged in repair of DNA lesions or in processes that allow cells to tolerate such lesions during DNA replication. Clonogenic assays show that brca1(-/-) mutants are 5-fold more sensitive to cisplatinum compared with wild-type cells. Our studies suggest that Brca1 contributes to damage repair and/or tolerance by promoting assembly of Rad51. This function appears to be shared with Brca2.

Animals↗

PubMed2000

Anti-angiogenic cues from vascular basement membrane collagen.

Vascular basement membrane is an important structural component of blood vessels and has been shown to interact with and modulate vascular endothelial behavior during angiogenesis. During the inductive phase of tumor angiogenesis, this membrane undergoes many degradative and structural changes and reorganizes to a native state around newly formed capillaries in the resolution phase. Such matrix changes are potentially associated with molecular modifications that include expression of matrix gene products coupled with conformational changes, which expose cryptic protein modules for interaction with the vascular endothelium. We speculate that these interactions provide important endogenous angiogenic and anti-angiogenic cues. In this report, we identify an important antiangiogenic vascular basement membrane-associated protein, the 26-kDa NC1 domain of the alpha1 chain of type IV collagen, termed arresten. Arresten was isolated from human placenta and produced as a recombinant molecule in Escherichia coli and 293 embryonic kidney cells. We demonstrate that arresten functions as an anti-angiogenic molecule by inhibiting endothelial cell proliferation, migration, tube formation, and Matrigel neovascularization. Arresten inhibits the growth of two human xenograft tumors in nude mice and the development of tumor metastases. Additionally, we show that the anti-angiogenic activity of arresten is potentially mediated via mechanisms involving cell surface proteoglycans and the alpha1beta1 integrin on endothelial cells. Collectively, our results suggest that arresten is a potent inhibitor of angiogenesis with a potential for therapeutic use.

Animals↗

PubMed2000

The role of cervical lymphadenectomy after aggressive concomitant chemoradiotherapy: the feasibility of selective neck dissection.

OBJECTIVES: To evaluate the necessity, technical feasibility, and complication rate of neck dissection performed on patients with head and neck cancer after 5 cycles of concomitant chemoradiotherapy (CRT) and to justify a selective neck dissection (SND) approach and define the optimal timing of post-CRT neck dissection. DESIGN AND SETTING: Retrospective analysis in an academic university medical center. PATIENTS: Sixty-nine eligible patients with advanced (stage III and IV) head and neck cancer who have undergone 1 of 4 CRT protocols. Patients ranged in age from 36 to 75 years, and surgical procedures were performed over a 4-year period. Follow-up ranged from 6 to 64 months. INTERVENTION: Neck dissection (most commonly unilateral SND) performed within 5 to 17 weeks after CRT completion. MAIN OUTCOME MEASURES: Complication rate and incidence of positive pathology (viable cancer) in pathologic neck dissection specimens. RESULTS: Seven (10%) of 69 patients developed wound healing complications, 4 (6%) of whom required surgical intervention for ultimate closure. There were no wound infections. Other complications occurred in 11 (16%) of 69 patients and included need for tracheotomy, nerve transection and paresis, and permanent hypocalcemia. Twenty-four (35%) of 69 patients revealed microscopic residual disease. Ten (50%) of 20 patients with N3 neck disease had positive pathology, whereas 14 (36%) of 39 patients with N2 disease had viable carcinoma in the dissection specimen (P =.09 by chi(2) analysis). There was no significant relation between radiologic complete response or partial response and residual microscopic cancer. In 1 patient, disease recurred in the neck after dissection. Mean follow-up time was 30.3 months. CONCLUSIONS: (1) Neck dissection for patients with N2 or greater neck disease after CRT is necessary to eradicate residual disease. (2) The complication rate of SND after CRT with hyperfractionated radiotherapy is low. (3) SNDs are technically feasible when performed within the "window" between the acute and chronic CRT injury (4-12 weeks). (4) SNDs, rather than more radical procedures, appear to be therapeutically appropriate in this group of patients because of the low incidence of disease recurrence in the neck.

Adult↗

PubMed2000

Gene therapy enhances the antiproliferative effect of radiation in intimal hyperplasia.

BACKGROUND: Although ionizing radiation (IR) has been demonstrated to attenuate vessel wall restenosis and intimal hyperplasia (IH), dose-related mural injury and atrophy are possible deleterious side effects. We tested the hypothesis that a radiosensitizing strategy may improve IR-induced inhibition of in vivo vascular smooth muscle cells (VSMCs) without influencing apoptotic cell death. METHODS: In 28 New Zealand White rabbits, the right common carotid artery (CCA) was injured and subjected to low-flow conditions to promote IH. The CCA was transfected with an adenoviral vector incorporating the cytosine deaminase (CD) gene (1 x 10(9) PFU/ml). 5-Fluorocytosine (5-FC), a prodrug that is converted to the radiosensitizing agent 5-fluorouracil (5-FU) by CD, was thereafter administered intravenously. The CCA was exposed to 5 Gy IR at 24 h. Intimal/medial (I/M) area and thickness ratios were determined in the harvested CCAs at 14 days. VSMC proliferative and apoptotic indices were assessed with immunohistochemistry. RESULTS: A 50% reduction in I/M area was found in rabbits treated with IR and IR + CD/5-FC (0.19 +/- 0.03 and 0.18 +/- 0.02) when compared with untreated controls (UC) (0.37 +/- 0.06) (P = 0.005). This finding was substantiated by attenuation of I/M thickness in the IR groups [0.47 +/- 0.13 (IR), 0.41 +/- 0.11 (IR + CD/5-FC), 0.61 +/- 0.17 (UC)] (P = 0.007). The number of proliferating VSMCs was notably smaller when IR was combined with CD/5-FC (4.17 +/- 1.16 vs 2.97 +/- 1.09 log transformed cells/mm(2), P < 0.07). Apoptosis was similar in all groups. CONCLUSIONS: Both IR alone and IR combined with a radiosensitizing agent are effective in attenuating experimental IH. However, combination therapy is synergistic and achieves greater inhibition of VSMC proliferation and may involve selective killing of radioresistant S-phase VSMCs. IR + CD/5-FC represents a novel therapeutic strategy that offers potential for long-term control of IH.

Animals↗

PubMed2000

Angiostatin induces mitotic cell death of proliferating endothelial cells.

Angiostatin is an inhibitor of tumor angiogenesis that induces regression of experimental tumors and enhances the antitumor effects of radiation therapy. We report that the cytotoxic effects of angiostatin are restricted to the proliferating endothelial cell population. In addition, angiostatin and ionizing radiation (IR) interact by inducing death of dividing endothelial cells. We also show that angiostatin and IR interact to inhibit endothelial cell migration. These findings demonstrate that angiostatin targets the proliferating tumor vasculature and provide a mechanistic basis for the cytotoxic interaction of angiostatin and IR.

Angiogenesis Inhibitors↗

PubMed2000

Evidence for the expression of radiation-induced potentially lethal damage being a p53-dependent process.

PURPOSE: To test the hypothesis that the expression of potentially lethal damage (PLD) is a p53-dependent process. MATERIALS AND METHODS: Previously reported data on radiation sensitivity, DNA double-strand break rejoining, PLD expression and repair (PLDR) were analyzed for a group of 12 human tumor cell lines and three human diploid fibroblast cell lines. Seven of these cell lines had normal p53 gene expression while the other eight were functionally p53-deficient. None of the cell lines was sensitive to radiation-induced apoptosis. RESULTS: Cell lines with a normal p53 expression were more sensitive to radiation, but only when sensitivity was measured in plateau-phase cultures under conditions where PLDR was minimized. Mutation or functional inactivation of p53 by HPV E6-transformation led to a more radioresistant phenotype under these conditions as well as a significant reduction in PLDR. PLDR was inversely proportional to the percentage of radiation-induced DNA double-strand breaks rejoined in 1 h in the p53 normal cell lines. CONCLUSIONS: These results suggest that the expression of PLD is primarily a p53-dependent process. In the absence of functional p53 gene expression, the effects of PLD are minimized. These observations help clarify the role of p53 in tumor response to radiation therapy because they suggest that the effects of alterations in p53 are highly dependent on the microenvironment of the tumor, i.e. whether conditions allow for PLDR.

Cell Line↗

PubMed2000

Down-regulation of ceramide production abrogates ionizing radiation-induced cytochrome c release and apoptosis.

Previous work has demonstrated that down-regulation of ceramide production after selection of cells with N-oleoylethanolamine (OE), an inhibitor of ceramidase, results in resistance to DNA damage-induced apoptosis. We report here that acute exposure of WEHI-231 cells (murine B-cell lymphoma) to OE activates neutral sphingomyelinase, induces ceramide production and increases intracellular reactive oxygen species. OE exposure also induces mitochondrial permeability, cytochrome c release, and apoptosis. Cells selected for resistance to OE exhibit little if any change in reactive oxygen species and cytochrome c release when exposed either to OE or to toxic doses of ceramide. Importantly, the OE resistant cells are also resistant to ionizing radiation-induced cytochrome c release and apoptosis. These findings demonstrate that down-regulation of neutral sphingomyelinase activity is associated with decreased DNA-damage-induced apoptosis. In addition, the data suggests that agents that modify extranuclear targets responsible for ceramide production select for cells resistant to ionizing radiation-induced apoptosis through alterations in mitochondrial function.

Animals↗

PubMed2000

E2F proteins are posttranslationally modified concomitantly with a reduction in nuclear binding activity in cells infected with herpes simplex virus 1.

The transition from G(1) to S phase in the cell cycle requires sequential activation of cyclin-dependent kinase 4 (cdk4) and cdk2, which phosphorylate the retinoblastoma protein, causing the release of E2F. Free E2F upregulates the transcription of genes involved in S phase and cell cycle progression. Recent studies from this and other laboratories have shown that herpes simplex virus 1 stabilizes cyclin D3 early in infection and that early events in viral replication are sensitive to inhibitors of some cdks. On the other hand cdk2 is not activated. Here we report studies on the status of members of E2F family in cycling HEp-2 and HeLa cells and quiescent serum-starved, contact-inhibited human lung fibroblasts. The results show that (i) at 8 h postinfection or thereafter, E2F-1 and E2F-5 were posttranslationally modified and/or translocated from nucleus to the cytoplasm, (ii) E2F-4 was hyperphophorylated, and (iii) overall, E2F binding to cognate DNA sites was decreased at late times after infection. These results concurrent with those cited above indicate that late in infection activation of S-phase genes is blocked both by posttranslational modification and translocation of members of E2F family to inactive compartments and by the absence of active cdk2. The observation that E2F were also posttranslationally modified in quiescent human lung fibroblasts that were not in S phase at the time of infection suggests that specific viral gene products are responsible for modification of the members of E2F family and raises the possibility that in infected cells, activation of the S phase gene is an early event in viral infection and is then shut off at late times. This is consistent with the timing of stabilization of cyclin D3 and the events blocked by inhibitors of cdks.

Alkaline Phosphatase↗

PubMed2000

Concomitant chemoradiotherapy as primary therapy for locoregionally advanced head and neck cancer.

PURPOSE: To achieve locoregional control of head and neck cancer, survival, and organ preservation using intensive concomitant chemoradiotherapy. PATIENTS AND METHODS: This study was a phase II trial of chemoradiotherapy with cisplatin 100 mg/m(2) every 28 days, infusional fluorouracil 800 mg/m(2)/d for 5 days, hydroxyurea 1 g orally every 12 hours for 11 doses, and radiotherapy twice daily at 1.5 Gy/fraction on days 1 through 5 (total dose, 15 Gy). Five days of treatment were followed by 9 days of rest, during which time patients received granulocyte colony-stimulating factor. Five cycles (three with cisplatin) were administered over 10 weeks (total radiotherapy dose, </= 75 Gy). Adjuvant chemoprevention with retinoic acid and interferon alfa-2A was offered. RESULTS: Seventy-six patients were treated (stage IV, 93%; N2, 54%; N3, 21%). At a median follow-up of 38 months, the 3-year progression-free survival is 72%, locoregional control 92%, systemic control 83%, and overall survival 55%. Toxicities included mucositis (grade 3, 45%; grade 4, 12%), neutropenia (grade 4, 39%), and thrombocytopenia (grade 4, 53%). Surgery at the primary site was performed in 13 patients, and 39 had neck dissection. A majority of patients declined adjuvant chemoprevention. Pharmacokinetic parameters were not prognostic of tumor control. Quality of life declined during treatment but returned from good to excellent by 12 months after treatment. CONCLUSION: Intensive concomitant chemoradiotherapy leads to high locoregional control and survival rates with organ preservation and a reversal of the historical pattern of failure (distant > locoregional). Surgery after concomitant chemoradiotherapy is feasible. Compliance with adjuvant chemoprevention is poor. Identification of less toxic regimens and improved distant disease control emerge as important future research goals.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2000

Surfactant sealing of membranes permeabilized by ionizing radiation.

Acute tissue injury and subsequent inflammation, including tissue edema and erythema, can be caused by sufficiently high levels of exposure to gamma radiation. The mechanism of this tissue injury is related to the generation of reactive oxygen intermediates (ROI) which chemically alter biological molecules and cell physiology. Cell membrane lipids are vulnerable to ROI-mediated lipid peroxidation that then leads to many of the acute tissue effects. We hypothesize that increased cell membrane permeability leading to osmotic swelling and vascular transudation is one of these effects. Thus we used adult postmitotic rhabdomyocytes in culture and microscopic fluorescence techniques to quantify radiation-induced changes in cell membrane permeability. Based on time-resolved dye flux measurements, a characteristic lag time of 34 +/- 3 min was determined between exposure to 160 Gy of gamma radiation and the decrease in membrane permeability. Administration of 0.1 mM nonionic surfactant Poloxamer 188 added to the cell medium after irradiation completely inhibited the dye loss over the time course of 2 h. Thus a reproducible model was developed for studying the mechanism of acute radiation injury and the efficacy of membrane-sealing agents. As only supportive measures now exist for treating the acute, nonlethal injuries from high-dose radiation exposure, agents that can restore cell membrane function after radiation damage may offer an important tool for therapy.

Animals↗

PubMed2000

The disappearance of cyclins A and B and the increase in activity of the G(2)/M-phase cellular kinase cdc2 in herpes simplex virus 1-infected cells require expression of the alpha22/U(S)1.5 and U(L)13 viral genes.

In uninfected cells the G(2)/M transition is regulated by cyclin kinase complex containing cdc2 and, initially, cyclin A, followed by cyclin B. cdc2 is downregulated through phosphorylation by wee-1 and myt-1 and upregulated by cdc-25C phosphatase. We have examined the accumulation and activities of these proteins in cells infected with wild type and mutants of herpes simplex virus 1. The results were as follows. (i) Cyclin A and B levels were reduced beginning 4 h after infection and were undetectable at 12 to 16 h after infection. (ii) cdc2 protein also decreased in amount but was detectable at all times after infection. In addition, a fraction of cdc2 protein from infected cells exhibited altered electrophoretic mobility in denaturing gels. (iii) The levels of cdk7 or myt-1 proteins remained relatively constant throughout infection, whereas the level of wee-1 was significantly decreased. (iv) cdc-25C formed novel bands characterized by slower electrophoretic mobility that disappeared after treatment with phosphatase. In addition, one phosphatase-sensitive band reacted with MPM-2 antibody that recognizes a phosphoepitope phosphorylated exclusively in M phase. (v) cdc2 accumulating in infected cells exhibited kinase activity. The activity of cdc2 was higher in infected cell lysates than those of corresponding proteins present in lysates of mock-infected cells even though cyclins A and B were not detectable in lysates of infected cells. (vi) The decrease in the levels of cyclins A and B, the increase in activity of cdc2, and the hyperphosphorylation of cdc-25C were mediated by U(L)13 and alpha22/U(S)1.5 gene products. In light of its normal functions, the activated cdc2 kinase may play a role in the changes in the morphology of the infected cell. These results are consistent with the accruing evidence that herpes simplex virus scavenges the cell for useful cell cycle proteins and subverts them for its own use.

CDC2 Protein Kinase↗

PubMed2000

In vitro and in vivo activity of protein kinase C inhibitor chelerythrine chloride induces tumor cell toxicity and growth delay in vivo.

Although clonogenic or divisional death is the main mechanism by which DNA-damaging agents demonstrate antitumor activity, recent data indicate that strategies specifically designed to trigger apoptosis may also prove to be useful antitumor agents. Protein kinase C (PKC) isoenzymes are involved in the regulation of cell proliferation, differentiation, and survival. Whereas pharmacological inhibition of PKC activity triggers apoptosis in most mammalian cells, cell line and tissue differences in sensitivities to these inhibitors remain. Whereas PKC inhibitors have potential as antitumor agents, issue of kinase specificity and solubility have remained obstacles to their clinical use. In this report, we investigated the antitumor activity of the PKC inhibitor chelerythrine chloride (chelerythrine), a selective inhibitor of group A and B PKC isoforms. Chelerythrine exhibited cytotoxic activity against nine human tumor cell lines tested in vitro. On the basis of the finding that radioresistant and chemoresistant squamous cell carcinoma lines (HNSCC) undergo apoptosis rapidly after treatment with chelerythrine in vitro, we assessed the effects of this agent on p53-deficient SQ-20B HNSCC cells in vivo. The results demonstrate that chelerythrine treatment of nude mice bearing SQ-20B is associated with significant tumor growth delay. Significantly, treatment with chelerythrine resulted in minimal toxicity. These findings demonstrate a potential for chelerythrine as an antitumor drug against squamous cell carcinoma.

Alkaloids↗