Ralf D. Hess

Co-expression of p53 and MDM2 in human atherosclerosis : Implications for the regulation of cellularity of atherosclerotic lesions

Atherosclerosis is a fibroproliferative disease of the arterial intima. It was recently found that wild‐type p53 (wt p53) accumulates in human atherosclerotic tissue. Wt p53 is a cell cycle regulator involved in DNA repair, DNA synthesis, cell differentiation, and apoptosis and might therefore make an important contribution to the cellularity of atherosclerotic plaques. The product of the MDM2 gene is a nuclear protein which forms a complex with p53, thereby inhibiting the negative regulatory effects of wt p53 on cell cycle progression. In order to address a potential role of the interaction of p53 with MDM2 for the regulation of cellularity in atherosclerotic tissue, 22 carotid atheromatous plaques from patients undergoing endarterectomy were studied to determine the presence of p53 immunoreactivity (IR), MDM2 IR, cell proliferation as evidenced by MIB1/Ki‐67 IR and DNA fragmentation by in situterminal transferase‐mediated dUTP 3′ end labelling (TUNEL), as a marker for apoptosis. p53 IR localized to areas with evidence of chronic inflammation (22/22) and was observed in virtually all cell types in 68·79±7·51 per cent of the nuclei. p53 staining in the control tissue from human internal mammary arteries was present in 0·2±0·29 per cent of the cells (P≤0·002). MDM2 IR was present in all cases (22/22) in macrophages and smooth muscle cells (SMCs) in 60·53±8·32 per cent of the nuclei (controls: 0·8±0·65 per cent, P≤0·002) and co‐localized with p53 IR as shown by examination of adjacent sections and by double immunofluorescence labelling. Importantly, co‐immunoprecipitation and western blot analysis revealed that p53 and MDM2 were physically associated, indicating that MDM2–p53 complex formation takes place in vivoin human atherosclerotic tissue. Positive TUNEL staining and MIB1/Ki‐67 IR present in 3·01±1·27 per cent of the nuclei (controls: 0 per cent, P≤0·002) localized to the same plaque compartments as p53 IR and MDM2 IR. Thus, the fate of cells with p53 accumulation may depend on the interaction and the stoichiometry of the p53 and MDM2 proteins. Cells were indeed found with strong p53 accumulation and nuclear morphology typical for apoptosis and there were a few MIB1/Ki‐67‐positive cells with co‐expression of MDM2, indicating a possible role for MDM2 in reversing the negative regulatory effects of p53 for cell cycle progression. The nuclear co‐localization of p53 IR with MDM2 IR and the co‐immunoprecipitation assay indicate the presence of p53–MDM2 complex formation in vivo in human atherosclerotic tissue. The destiny of individual p53 and MDM2‐co‐expressing cells either to undergo p53‐dependent apoptosis or to re‐enter the cycle of cell proliferation may depend on the relative ratios of the two proteins. p53 and MDM2 may therefore play an important role in regulating cellularity and inflammatory activity in human atherosclerotic plaques.

Regulatory, biosafety and safety challenges for novel cells as substrates for human vaccines.

In the development of novel substrates used for production of human vaccines there has been significant progress made in recent years. Emerging and re-emerging infectious diseases like the recent porcine Influenza A virus (H1N1) pandemic necessitated the availability of unprecedented amounts of vaccines. In addition, the high demand for vaccines in the industrialised countries has also been paralleled by a steep increase in demand in developing countries. The manufacturing capability for viral vaccines produced in embryonated hen eggs and conventional/classical cell substrates, such as chicken embryo fibroblasts, has now reached its capacity limit. This constraint may be overcome by utilising other recognised cell substrates such as Madin Darby Canine Kidney (MDCK) (dog origin), Chinese Hamster Ovary (CHO) (hamster cells) or Vero cells (monkey origin) or as an alternative, introduce new cell substrates of human or avian origin. Using new cell substrates may prove to be a highly replication-proficient way of producing live viral vaccines such as Influenza A viruses. Despite some advantages, cell substrates may pose a small residual risk to humans since some of them are known to be tumourigenic in immunosuppressed animals. However, this residual risk should be considered acceptable by regulators. Safety testing requirements for cell substrates used in the manufacture of vaccines is mandated by published guidance from organisations such as World Health Organization (WHO), United States Food and Drug Administration (FDA), European Medicines Agency (EMA) and International Conferences on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human use (ICH) as well as requirements laid down in compendial monographs (Ph. Eur. and USP). This paper considers the guidance contained in these regulatory documents. In addition, the safety challenges and almost arbitrary risk-based classification of cell substrates used in the production of human vaccines together with compliance to GCCP (Good Cell Culture Practice) are discussed. Even though there has been tremendous progress in the last few years, reflected mainly by revisions and updates to regulatory guidance documents, there still is still no consensus between regulators nor significant harmonisation of the guidance documents or monographs.

Nuclear accumulation of p53 in response to treatment with DNA-damaging agents

A number of agents which damage DNA also trigger the nuclear accumulation of the tumor suppressor protein p53. Here we show the correlation with different p53 detection methods. As an example we investigated the effects of the cancer therapy drug mitomycin C on different mammalian cell lines. Our findings demonstrate that either the immunofluorescence techniques (indirect immunofluorescence staining or flow cytometric analysis) or ELISA or immunoblot assays are useful methods in detecting p53 accumulation. Simultaneously we measured DNA damage with the terminal deoxynucleotidyl transferase assay. Compatible data were obtained. Thus p53 accumulation may be used as indicator of DNA injury.

DNA-damage-inducible p53 activity in SV40-transformed cells.

The biological state of the tumour suppressor proteins Rb and p53 is altered in papillomavirus- and SV40-transformed cells, due to interaction with the DNA tumour virus oncogene proteins E6/E7 and the tumour (T) antigen. Thus, the DNA damage response function of p53, a crucial feature of the tumour suppressor p53, might be considered as inactive. To investigate this subject, C57SV and VLM, two SV40-transformed murine cell lines enharboring constitutively high nuclear p53 and SV40 large T antigen levels, were treated with mitomycin C. Mitomycin C is known for its activity to elicit DNA damage, followed by nuclear accumulation of biologically active p53. Surprisingly, the nuclear p53 level significantly increased in mitomycin-C-treated C57SV cells and to a lesser degree in VLM cells. In addition, expression of p21WAF1 protein was induced in C57SV and VLM cells. This indicates a possible DNA-damage-elicited p53 activation. Finally, nuclear extracts of mitomycin-C-treated C57SV and VLM cells, but not of untreated cells, exhibited PAb421-enhanced specific DNA-binding activity of p53, as proven by gel shift analysis. Thus, DNA damage induced essential biological functions typical for wild-type p53 in the SV40-transformed cell lines examined so far.

Protection of mice against SV40 tumours by Pam3Cys, MTP-PE and Pam3Cys conjugated with the SV40 T antigen-derived peptide, K(698)-T(708).

The intraperitoneal injection of Balb/c mice with synthetic analogues of adjuvants S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-R-cysteine (Pam3Cys) or muramyltripeptide phosphatidylethanolamine (MTP-PE) inhibited the tumourigenic growth of subcutaneously injected VLM cells, a syngeneic simian virus 40 (SV40)-transformed cell line. Furthermore, the Pam3Cys conjugate of K698-T708 (KT), which represents the C-terminal undecapeptide of the SV40 large tumour (T) antigen, was tumour-protective. Also syngeneic spleen cells, preincubated in vitro with this Pam3Cys-KT derivative, which anchores spontaneously at the cell membrane, were, through SV40 tumour mimicry, tumour-protective. The protection was impaired by treatment of the mice with either anti-CD4, anti-CD8 IgG, anti asialo GM1 antiserum or dextrane sulfate, which deplete the CD4+, CD8+ and NK cells or the macrophages, respectively. In summary, SV40 tumour transplantation resistance can be experimentally elicited by a tumour-epitope-specific vaccine. In the absence of an immunogenic epitope protection was obtained by administration of biological response modifiers. Protection is effected by SV40-T-antigen-specific cytotoxic lymphocytes in cooperation with NK cells and macrophages.

rhG-CSF affects genes involved in mitogen signalling and early gene expression in the ovarian cancer cell line HEY

The ovarian adenocarcinoma cell line HEY was used as an in vitro model to study the influence of recombinant human granulocyte colony‐stimulating factor (rhG‐CSF) on epithelial tumours such as ovarian cancer. Serum‐starved cells were treated with rhG‐CSF in a time‐ and dose‐dependent manner. Cell proliferation, measured as cell division and DNA synthesis, was stimulated about 40% by rhG‐CSF. After harvesting, cells were examined for the presence of G‐CSF receptor (FACS analysis and RT‐PCR), as well as for expression of genes involved in mitogen signalling (ERKs, JNKs) and early gene expression (c‐jun). rhG‐CSF affected mitogen‐activated pathways and was receptor‐mediated if the G‐CSF receptor was present. After rhG‐CSF induction, Janus N‐terminal kinases (JNK 1 and 2) were simultaneously increased in the cytosol, up to 30‐fold as measured by Western blotting), whereas ERK 1 and 2 accumulated maximally by 2.5‐fold 1 hr after rhG‐CSF induction. c‐Jun was up‐regulated strongly by this cytokine at the translational level. Our data suggest that rhG‐CSF affects genes involved in mitogen signalling and early gene expression in solid tumours. We also noted the presence of G‐CSF receptor on ovarian cancer cell lines. Int. J. Cancer 75:847–854, 1998.

DNA damage by filtered, tar- and aerosol-free cigarette smoke in rodent cells: a novel evaluation

Nuclear accumulation of the tumorsuppressor protein p53 indicates the occurrence of chromatin injury (J. Cancer Res. Clin. Oncol. 1991, 117, 30; Oncogene 1993, 8, 307) and may be used as an analytical tool to detect genotoxic agents. This mechanism was used to evaluate the DNA-damaging potency (clastogenicity) of the tar- and aerosol-free, gaseous phase of cigarette smoke which is obtained by filtration through Cambridge glass fiber filters. This condensate-free gas phase was absorbed by phosphate-buffered saline and immediately thereafter poured onto monolayers of the murine cell line L929 for 10 min. Eighteen hours later the nuclear accumulation of p53, an indicator for DNA damage, was determined. The elicited level of p53 was similar to that obtained by direct incubation with the gas phase of filtered cigarette smoke for 2 min or with several micrograms of mitomycin C per ml. Previous exhaustive filtration obviously does not inhibit the clastogenic property of tobacco smoke to exert severe DNA damage.

The cell-binding carboxyterminal undecapeptide of SV40 tumour antigen provides protective cell-dependent immunity

This paper describes the use of the synthetic carboxyterminal undecapeptide of large SV40 tumour antigen, lys698-thr708 (KT) to protect Balb/c mice against growth of subcutaneously transplanted tumorigenic SV40-transformed cells (VLM). The vaccine was prepared by conjugation of KT with 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide (SPDP). Addition of the SPDP-derivative of KT to syngeneic spleen cells rendered KT covalently linked to free thiol-groups of the cell membranes by the formation of -S-S-CH2-CH2-CO-epsilon-NH-lys698 bonds. Vaccination with KT-conjugated cells was intraperitoneal. Alternatively, KT-conjugated cells were generated in the peritoneum by injection of PDP-KT ((2-pyridyldithio)propionic acid-KT). As a control 60Co-irradiated VLM cells were used. In five experiments all VLM-vaccinated and the majority of the PDP-KT-(or KT-spleen cell)-vaccinated mice were protected against tumour growth. However, mice pretreated with saline, unconjugated spleen cells, free KT, KT conjugated to bovine serum albumin, or KT with incomplete Freunds adjuvant developed tumours. Treatment of PDP-KT-vaccinated mice with anti-CD4 or anti-CD8 immunoglobulin abolished tumour immunity completely. Thus, covalent binding of the carboxyterminal undecapeptide of SV40 tumour antigen to viable, untransformed cells yielded a vaccine which protects Balb/c mice against SV40 tumours.

Monospecific polyclonal anti-anti-idiotypic antibodies to the carboxyterminal undecapeptide of the SV40 large tumour antigen.

The murine monoclonal antibody PAb1605 defines an epitope, peptide Lys(698)‐Thr(708) (KT), on the carboxyterminus of the tumour(T)antigen of SV40‐transformed cells. In vivo and in vitro experiments had shown that this sequence represents an epitope for both humoral and cellular immune responses. When injected into rabbits PAb1605 induces anti‐idiotypic antibodies (Ab‐2). Ab‐2β (internal image type) was purified by adsorption chromatography and characterized by the ability of KT to compete with the binding of ab‐2 with ab‐1. Murine anti‐anti‐idiotypic antibodies (ab‐3) were obtained by immunization of mice with ab‐2β. Both ab‐1 and ab‐3 JgG showed affinities to immunoprecipitated SV40 T antigen by immunoblot analysis and to nuclear SV40 T antigen by the immunofluorescence assay. The binding of ab‐3 to SV40 T antigen was completely inhibited by competition with KT. We conclude that the polyclonal ab‐3 is of the ab‐3 subtype and specific for only one epitope which is represented by KT and defined by ab‐1. The results demonstrate that the specificity for a defined peptide epitope of an antibody was conserved even after two consecutive steps of anti‐idiotypic‐antibody formation in two host species. Since this postulate of network theory could be verified for a sequence of a tumour‐associated antigen which represents a B‐ and T cell epitope, this model is of great interest for further tumour immunological studies.

PAb1614, a Monoclonal Antibody Reactive with the Tumor Antigens of SV40, JC, BK, and Polyoma Virus, and Other JC Virus Tumor Antigen Cross-Reactive Antibodies of the PAb1601-1636 Panel

PAb1614, an SV40-specific monoclonal antibody of the panel PAb1601-1636 reacts with large and small tumor antigens of SV40, BK and JC virus, and with polyoma virus large and middle tumor antigens, but not with the large tumor antigen of the lymphotropic papova virus. Using immunofluorescence and immunoblot competition assays and ELISA with synthetic peptides, it is shown that the epitope is represented by the SV40 tumor antigen undecapeptide, K39-E49. This peptide comprises the tumor antigen consensus sequence, H42-G47, of the polyoma viruses. However, the epitope of PAb1614 probably does not exactly coincide with this hexapeptide. This explains why some cross-reactions are less strong, or absent, as in the case of the lymphotropic papova virus. Further antibodies of the PAb1601-1636 panel that cross-react with the JC virus large tumor antigen are PAb1602, 1604, 1606, 1618, 1621, 1622, 1623, 1624, 1626, 1629, and 1633.