Josef Michl

Selective binding of a monoclonal antibody to Aspergillus niger glucose oxidase by formaldehyde fixed human polymorphonuclear leukocytes.

BACKGROUND Many of the procedures used in handling neutrophils may affect the expression of surface antigens, and hence their quantitation by flow cytometry. METHODS Because the enzyme glucose oxidase of Aspergillus niger is absent in human tissues, an IgM against it (mAb GO) was used as negative control in a study involving the normal expression of neutrophil specific BH2-Ag in different age groups. RESULTS When peripheral blood leukocytes (PBL) were freshly prepared, processed and stained with FITC-mAb GO without fixation or when the cells were stained with FITC-mAb GO prior to fixation with 2% formaldehyde, both median fluorescent intensity (MFI) and per cent of positively stained polymorphonuclear leukocytes (PMN) were similar to that obtained with a background sample without any antibody. However, when PBL were fixed after isolation with different concentrations of formaldehyde and for varying durations, MFI and per cent of positively stained PMN but not of monocytes or lymphocytes with FITC-mAb GO increased in a time and concentration dependent manner. Saturation was achieved at a finite concentration of the antibody. In a competition assay unlabelled mAb GO reduced binding of FITC-mAb GO to PMN by 79% and 95% at concentrations 100 and 200 times that of FITC labelled antibody, respectively. CONCLUSIONS These observations strongly suggest that formaldehyde fixation causes the expression or accessibility of an epitope on PMN that is specifically recognized by the mAb GO.

Abstract 729: Mechanism of action of a p53-derived peptide that targets a novel death pathway inducing selective necrosis in cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC In a series of preliminary in vitro and in vivo studies we have shown that p53-derived peptides consisting of residues 12-26 and 17-26 from the murine double minute binding domain (MDM-2) possess anti-cancer activity. These peptides are linked to a trans-membrane penetrating sequence (penetratin) derived from the antennapedia homeodomain of Drosophila and are designated PNC-27 and PNC-28, respectively. We also found that the previously determined 3D structure of these PNC residues are directly superimposeable on the same residues bound to MDM-2, suggesting that these peptide may target MDM-2 in cancer cells. These peptide constructs appear to promote cancer cell death through a novel pathway that leads to necrosis rather than apoptosis while sparing their untransformed counterparts. In our initial experiments, the critical role of the MDM-2 oncoprotein as a potential target molecule for these PNC peptides was elucidated when a sequence specific MDM-2 antibody blocked PNC-27 activity on cancer cells. To further elucidate this anti-cancer mechanism we then tested parent molecule PNC-27 against selected cancer cell lines (Panc-02, pancreatic; HT1080, fibrosarcoma; and MCF-7, breast) plus respective untransformed counterparts (BMRPA1, BJ, and MCF-10) which were treated with PNC-27 and control peptide. Subsequently, treatment with fluorescent labeled peptide, immunoprecipitation (IP), confocal, and time-lapse electron microscopy (EM) was employed as well as transfection of untransformed cells with the MDM-2 gene to study the anti-cancer mechanism. PNC-27 did not cause elevation of pro-apoptotic proteins but induced rapid dose-dependent tumor cell death with release of lactate dehydrogenase (LDH). This specific anti-cancer mechanism, in part, may be due to observed increased expression of MDM-2 found in cancer cells compared to their normal cellular counterparts which is consistent with abrogation of MDM-2 and stabilization of p53 protein levels after PNC-27 treatment. Moreover, following IP of fluorescent labeled PNC-27 from treated cancer cells; labeled MDM-2 was detected. Furthermore, confocal microscopy and EM revealed co-localization of PNC-27 and MDM-2 along with cancer cell membrane pore formation, respectively. Remarkably, transfection of the MDM-2 gene resulted with increased expression of MDM-2 and now untransformed cell susceptibility to PNC-27. These results suggest a mechanism of PNC-27 binding to MDM-2 in the cancer cell membrane, leading to pore formation and membrane disruption; a p53-independent pathway consistent with rapid selective cancer cell necrosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 729.

Abstract 5770: MDM2 protein variants expression in the plasma membrane of cancer cells: A target for anticancer peptide PNC-27

PNC-27 anti-cancer peptide, derived from the MDM2 binding site of p53 and linked to a membrane residency peptide (MRP), has been shown to cause necrosis of cancer cells without affecting untransformed cells. PNC-27 was also able to eradicate pancreatic tumor xenografts in mice (Michl et al, Int. J of Cancer, 2006). Recently, we have identified the mechanism of action of this peptide as due to formation of oligomeric pores in the plasma membrane (PM) of cancer cells but not in the PM of untransformed cells. The mechanism of pore-formation by PNC-27 closely resembles the pore-formation process by streptolysin-O, melittin and similar pore-forming agents. We have shown MDM2 as a targeting molecule that leads to PNC-27 selectivity towards cancer cells by its mis-localization to cancer cell PM (Sarafraz-Yazdi et al, PNAS, in press). Examining purified PM of a variety of cancer cells by immunoblotting, we now provide evidence for multiple MDM2 protein variants, and that were absent in the PM of normal untransformed cells. To confirm the purity of the isolated PM, fractions were also immunoblotted for the PM markers Na+/K+-ATPase, E-Cadherin and s-Catenin, all of which were enriched in the PM fractions. In contrast, Abs against membrane markers specific for intracellular organelles, COX IV and Cytochrome C for mitochondrial membrane, showed no reaction in the PM fractions while they reacted with total membrane fractions. Three protein variants of MDM2 with MW of 27kD, 40kD and 57kD were consistently expressed in the PM of human and rat pancreatic cancer cells, human melanoma, breast cancer. We also confirmed these observations in isolated PM from freshly obtained primary ovarian tumors from human patients with aggressive tumor. Remarkably, no MDM2 variant was detectable in the PM fractions of primary human fibroblasts and untransformed pancreatic HPNE cells. Our finding of differently sized MDM2 variants complies with other, previously reported MDM2 protein variants. Of the different MDM2 mRNA splice variants published, 5 have been shown to be translated into protein including proteins of 27kD, 40kD and 57kD. Our present study demonstrates the localization of the 3 variant proteins in the PM of cancer cells, suggesting a possible role for these membrane-localized MDM2 variant in the action of PNC-27. The notion of this specific MDM2-PNC-27 interaction at the PM level was strongly supported by the effective competition by a monoclonal MDM2-specific Ab, reducing PNC-27-mediated cytotoxicity by >80% as measured by LDH cytotoxicity assay and propidium iodide staining of the nuclei of dead cells in real-time spinning disc confocal microscopy. These findings for the first time demonstrate not only the novel localization of different MDM2 variants in PM of different cancer cells but they also suggest a potentially wider applicability of PNC-27 as a novel selective anti-cancer drug for clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5770.