Konstantin Christov

Dual effect of p53 on radiation sensitivity in vivo: p53 promotes hematopoietic injury, but protects from gastro-intestinal syndrome in mice

Ionizing radiation (IR) induces p53-dependent apoptosis in radiosensitive tissues, suggesting that p53 is a determinant of radiation syndromes. In fact, p53-deficient mice survive doses of IR that cause lethal hematopoietic syndrome in wild-type animals. Surprisingly, p53 deficiency results in sensitization of mice to higher doses of IR, causing lethal gastro-intestinal (GI) syndrome. While cells in the crypts of p53-wild-type epithelium undergo prolonged growth arrest after irradiation, continuous cell proliferation ongoing in p53-deficient epithelium correlates with accelerated death of damaged cells followed by rapid destruction of villi and accelerated lethality. p21-deficient mice are also characterized by increased sensitivity to GI syndrome-inducing doses of IR. We conclude that p53/p21-mediated growth arrest plays a protective role in the epithelium of small intestine after severe doses of IR. Pharmacological inhibition of p53 by a small molecule that can rescue from lethal hematopoietic syndrome has no effect on the lethality from gastro-intestinal syndrome, presumably because of a temporary and reversible nature of its action.

Different impact of p53 and p21 on the radiation response of mouse tissues

Mammalian tissues differ dramatically in their sensitivity to genotoxic stress, although the mechanisms determining these differences remain largely unknown. To analyse the role of p53 and p21 in determination of tissue specificity to DNA damage in vivo, we compared the effects of γ radiation on DNA synthesis on whole-body sections of wild type, p53-deficient and p21-deficient mice. A dramatic reduction in 14C-thymidine incorporation after γ irradiation was observed in the majority of rapidly proliferating tissues of wild type and p21−/− but not in p53−/− mice, confirming the key role of p53 in determination of tissue response to genotoxic stress in vivo and suggesting that p53-mediated inhibition of DNA synthesis does not depend on p21. Rapid radiation induced p53-dependent apoptosis was mapped to the areas of high levels of p53 mRNA in radiation sensitive tissues analysed (white pulp in the spleen and bases of crypts in small intestine), indicating that p53 regulation at the mRNA level is a determinant of cellular sensitivity to genotoxic stress. High p53 mRNA expression is inherited as a recessive trait in cell–cell hybrids suggesting the involvement of a negative control mechanism in the regulation of p53 gene expression.

Bacterial cupredoxin azurin as an inducer of apoptosis and regression in human breast cancer

Azurin, a copper-containing redox protein released by the pathogenic bacterium Pseudomonas aeruginosa, is highly cytotoxic to the human breast cancer cell line MCF-7, but is less cytotoxic toward p53-negative (MDA-MB-157) or nonfunctional p53 cell lines like MDD2 and MDA-MB-231. The purpose of this study was to investigate the underlying mechanism of the action of bacterial cupredoxin azurin in the regression of breast cancer and its potential chemotherapeutic efficacy. Azurin enters into the cytosol of MCF-7 cells and travels to the nucleus, enhancing the intracellular levels of p53 and Bax, thereby triggering the release of mitochondrial cytochrome c into the cytosol. This process activates the caspase cascade (including caspase-9 and caspase-7), thereby initiating the apoptotic process. Our results indicate that azurin-induced cell death stimuli are amplified in the presence of p53. In vivo injection of azurin in immunodeficient mice harboring xenografted human breast cancer cells in the mammary fat pad leads to statistically significant regression (85%, P=0.0179, Kruskal–Wallis Test) of the tumor. In conclusion, azurin blocks breast cancer cell proliferation and induces apoptosis through the mitochondrial pathway both in vitro and in vivo, thereby suggesting a potential chemotherapeutic application of this bacterial cupredoxin for the treatment of breast cancer.

Noncationic Peptides Obtained From Azurin Preferentially Enter Cancer Cells

Azurin, a member of the cupredoxin family of copper containing redox proteins, preferentially penetrates human cancer cells and exerts cytostatic and cytotoxic (apoptotic) effects with no apparent activity on normal cells. Amino acids 50 to 77 (p28) of azurin seem responsible for cellular penetration and at least part of the antiproliferative, proapoptotic activity of azurin against a number of solid tumor cell lines. We show by confocal microscopy and fluorescence-activated cell sorting that amino acids 50 to 67 (p18) are a minimal motif (protein transduction domain) responsible for the preferential entry of azurin into human cancer cells. A combination of inhibitors that interfere with discrete steps of the endocytotic process and antibodies for caveolae and Golgi-mediated transport revealed that these amphipathic, alpha-helical peptides are unique. Unlike the cationic cell-penetrating peptides, alpha-helical antennapedia-like, or VP22 type peptides, p18 and p28 are not bound by cell membrane glycosaminoglycans and preferentially penetrate cancer cells via endocytotic, caveosome-directed, and caveosome-independent pathways. Once internalized, p28, but not p18, inhibits cancer cell proliferation initially through a cytostatic mechanism. These observations suggest the azurin fragments, p18 and p28, account for the preferential entry of azurin into human cancer cells and a significant amount of the antiproliferative activity of azurin on human cancer cells, respectively.

Hemizygous Disruption of Cdc25A Inhibits Cellular Transformation and Mammary Tumorigenesis in Mice

CDC25A phosphatase activates multiple cyclin-dependent kinases (CDK) during cell cycle progression. Inactivation of CDC25A by ubiquitin-mediated degradation is a major mechanism of DNA damage-induced S-G(2) checkpoint. Although increased CDC25A expression has been reported in various human cancer tissues, it remains unclear whether CDC25A activation is a critical rate-limiting step of carcinogenesis. To assess the role for CDC25A in cell cycle control and carcinogenesis, we used a Cdc25A-null mouse strain we recently generated. Whereas Cdc25A(-/-) mice exhibit early embryonic lethality, Cdc25A(+/-) mice show no appreciable developmental defect. Cdc25A(+/-) mouse embryonic fibroblasts (MEF) exhibit normal kinetics of cell cycle progression at early passages, modestly enhanced G(2) checkpoint response to DNA damage, and shortened proliferative life span, compared with wild-type MEFs. Importantly, Cdc25A(+/-) MEFs are significantly resistant to malignant transformation induced by coexpression of H-ras(V12) and a dominant negative p53 mutant. The rate-limiting role for CDC25A in transformation is further supported by decreased transformation efficiency in MCF-10A human mammary epithelial cells stably expressing CDC25A small interfering RNA. Consistently, Cdc25A(+/-) mice show substantially prolonged latency in mammary tumorigenesis induced by MMTV-H-ras or MMTV-neu transgene, whereas MMTV-myc-induced tumorigenesis is not significantly affected by Cdc25A heterozygosity. Mammary tissues of Cdc25A(+/-);MMTV-neu mice before tumor development display less proliferative response to the oncogene with increased tyrosine phosphorylation of CDK1/2, but show no significant change in apoptosis. These results suggest that Cdc25A plays a rate-limiting role in transformation and tumor initiation mediated by ras activation.

Deregulated CDC25A Expression Promotes Mammary Tumorigenesis with Genomic Instability

Checkpoint pathways help cells maintain genomic integrity, delaying cell cycle progression in response to various risks of fidelity, such as genotoxic stresses, compromised DNA replication, and impaired spindle control. Cancer cells frequently exhibit genomic instability, and recent studies showed that checkpoint pathways are likely to serve as a tumor-suppressive barrier in vivo. The cell cycle-promoting phosphatase CDC25A is an activator of cyclin-dependent kinases and one of the downstream targets for the CHK1-mediated checkpoint pathway. Whereas CDC25A overexpression is observed in various human cancer tissues, it has not been determined whether deregulated CDC25A expression triggers or promotes tumorigenesis in vivo. Here, we show that transgenic expression of CDC25A cooperates markedly with oncogenic ras or neu in murine mammary tumorigenesis. MMTV-CDC25A transgenic mice exhibit alveolar hyperplasia in the mammary tissue but do not develop spontaneous mammary tumors. The MMTV-CDC25A transgene markedly shortens latency of tumorigenesis in MMTV-ras mice. The MMTV-CDC25A transgene also accelerates tumor growth in MMTV-neu mice with apparent cell cycle miscoordination. CDC25A-overexpressing tumors, which invade more aggressively, exhibit various chromosomal aberrations on fragile regions, including the mouse counterpart of human 1p31-36, according to array-based comparative genomic hybridization and karyotyping. The chromosomal aberrations account for substantial changes in gene expression profile rendered by transgenic expression of CDC25A, including down-regulation of Trp73. These data indicate that deregulated control of cellular CDC25A levels leads to in vivo genomic instability, which cooperates with the neu-ras oncogenic pathway in mammary tumorigenesis.

A first-in-class, first-in-human, phase I trial of p28, a non-HDM2-mediated peptide inhibitor of p53 ubiquitination in patients with advanced solid tumours

Background:This first-in-human, phase I clinical trial of p28 (NSC745104), a 28-amino-acid fragment of the cupredoxin azurin, investigated the safety, tolerability, pharmacokinetics and preliminary activity of p28 in patients with p53+ metastatic solid tumours.Methods:A total of 15 patients were administered p28 i.v. as a short infusion three times per week for 4 weeks followed by a 2-week rest under an accelerated titration 3+3 dose escalation design until either a grade 3-related adverse event occurred or the maximum tolerated dose (MTD) was reached. Single-dose and steady-state serum pharmacokinetics were characterised. Assessments included toxicity, best objective response by RECIST 1.1 Criteria, and overall survival.Results:No patients exhibited any dose-limiting toxicities (DLTs), significant adverse events or exhibited an immune response (IgG) to the peptide. The No Observed Adverse Effect Level (NOAEL) and MTD were not reached. Seven patients demonstrated stable disease for 7–61 weeks, three a partial response for 44–125 weeks, and one a complete response for 139 weeks. Three patients are still alive at 158, 140, and 110 weeks post therapy completion.Conclusion:p28 was tolerated with no significant adverse events. An MTD was not reached. Evidence of anti-tumour activity indicates a highly favourable therapeutic index and demonstrates proof of concept for this new class of non-HDM2-mediated peptide inhibitors of p53 ubiquitination.

A peptide fragment of azurin induces a p53-mediated cell cycle arrest in human breast cancer cells

We report that amino acids 50 to 77 of azurin (p28) preferentially enter the human breast cancer cell lines MCF-7, ZR-75-1, and T47D through a caveolin-mediated pathway. Although p28 enters p53 wild-type MCF-7 and the isogenic p53 dominant-negative MDD2 breast cancer cell lines, p28 only induces a G2-M-phase cell cycle arrest and apoptosis in MCF-7 cells. p28 exerts its antiproliferative activity by reducing proteasomal degradation of p53 through formation of a p28:p53 complex within a hydrophobic DNA-binding domain (amino acids 80-276), increasing p53 levels and DNA-binding activity. Subsequent elevation of the cyclin-dependent kinase inhibitors p21 and p27 reduces cyclin-dependent kinase 2 and cyclin A levels in a time-dependent manner in MCF-7 cells but not in MDD2 cells. These results suggest that p28 and similar peptides that significantly reduce proteasomal degradation of p53 by a MDM2-independent pathway(s) may provide a unique series of cytostatic and cytotoxic (apoptotic) chemotherapeutic agents. [Mol Cancer Ther 2009;8(10):2947–58]

Short-term Modulation of Cell Proliferation and Apoptosis and Preventive/Therapeutic Efficacy of Various Agents in a Mammary Cancer Model

Purpose: The methylnitrosourea (MNU)-induced mammary cancer model in rats is similar to estrogen receptor–positive breast cancer in women. In prevention studies using this model, tumor incidence and multiplicity were typically primary end points. The ability of various agents administered for a short period to modulate cell proliferation [proliferation index (PI)] and apoptosis [apoptotic index (AI)] in mammary cancers was compared with their efficacy in long-term prevention and therapy studies. Experimental Design: Rats were injected with MNU to induce mammary cancers. For the prevention studies, agents were administered by gavage or in the diet beginning 5 days after MNU. For proliferation (PI) and apoptosis (AI) experiments, animals with a palpable mammary cancer were treated with the agents for only 4 to 7 days. PI was determined following 5-bromodeoxyuridine labeling whereas AI was determined using the terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling assay. Therapeutic efficacy was evaluated by measuring cancer size over a 6-week period. Results: Treatments with differing chemopreventive efficacy and mechanism(s) of action were examined: (a) hormonal treatments [tamoxifen, vorozole (an aromatase inhibitor), and ovariectomy]; (b) retinoid X receptor agonists (targretin, 9-cis retinoic acid, and UAB30); (c) inducers of drug-metabolizing enzymes (indole-3-carbinol, 5,6 benzoflavone, and diindoylmethane); (d) agents that alter signal transduction (R115777, a farnesyltransferase inhibitor); Iressa (an epidermal growth factor receptor inhibitor); sulindac and celecoxib (cyclooxygenase 1/2 and cyclooxygenase 2 inhibitors); and (e) diverse agents including meclizine, vitamin C, and sodium phenylbutyrate. Correlations between inhibition of PI, increase of AI, and chemopreventive efficacy were observed. Although most agents with moderate or low preventive efficacy suppressed PI, they minimally affected AI. Conclusions: The data confirmed that the short-term effects of various agents on cell proliferation and apoptosis in small mammary cancers can predict their preventive/therapeutic efficacy. Thus, these biomarkers can be used to help determine the efficacy of compounds in phase II clinical prevention trials.

Multiple Myeloma Regression Mediated by Bruceantin

Purpose: Bruceantin has been shown to induce cell differentiation in a number of leukemia and lymphoma cell lines. It also down-regulated c-MYC, suggesting a correlation of down-regulation with induction of cell differentiation or cell death. In the present study, we focused on multiple myeloma, using the RPMI 8226 cell line as a model. Experimental Design: The effects of bruceantin on c-MYC levels and apoptosis were examined by immunoblotting, 4′,6-diamidino-2-phenylindole staining, evaluation of caspase-like activity, and 3,3′-dihexyloxacarbocyanine iodide staining. The potential of bruceantin to inhibit primary tumor growth was assessed with RPMI 8226 xenografts in SCID mice, and apoptosis in the tumors was evaluated by the terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Results: c-MYC was strongly down-regulated in cultured RPMI 8226 cells by treatment with bruceantin for 24 h. With U266 and H929 cells, bruceantin did not regulate c-MYC in this manner. Apoptosis was induced in the three cell lines. In RPMI 8226 cells, apoptosis occurred through proteolytic processing of procaspases and degradation of poly(ADP-ribose) polymerase. The mitochondrial pathway was also involved. Because RPMI 8226 cells were the most sensitive, they were used in a xenograft model. Bruceantin treatment (2.5–5 mg/kg) resulted in a significant regression of tumors without overt toxicity. Apoptosis was significantly elevated in tumors derived from animals treated with bruceantin (37%) as compared with the control tumors (14%). Conclusions: Bruceantin interferes with the growth of RPMI 8226 cells in cell culture and xenograft models. These results suggest that bruceantin should be reinvestigated for clinical efficacy against multiple myeloma and other hematological malignancies.