Bernd Kaina

Rho GTPases are over-expressed in human tumors.

Small GTPases of the Rho family are involved in the regulation of a variety of cellular processes, such as the organization of the microfilamental network, cell‐cell contact and malignant transformation. To address the question of whether Rho proteins are involved in carcinogenesis in man, we compared their expression in tumors from colon, breast and lung with that of the corresponding normal tissue originating from the same patient. As shown by Rho‐specific 32P‐ADP‐ribosylation, as well as Western‐blot analysis, the amount of RhoA protein was largely increased in all 3 types of tumors tested. The most dramatic differences in the expression of Rho GTPases were observed in breast tissue. All breast tumors analyzed showed high levels of RhoA, Rac and Cdc42 proteins, whereas in the corresponding normal tissue these Rho proteins were hardly or not detectable. Progression of breast tumors from WHO grade I to grade III was accompanied by a significant average increase in RhoA protein. Overall, increase in the amount of Rho GTPases, in particular RhoA, appears to be a frequent event in different types of human tumors. This supports the view that Rho GTPases are involved in human carcinogenesis. Int. J. Cancer 81:682–687, 1999.

Mechanisms of human DNA repair: an update.

The human genome, comprising three billion base pairs coding for 30000-40000 genes, is constantly attacked by endogenous reactive metabolites, therapeutic drugs and a plethora of environmental mutagens that impact its integrity. Thus it is obvious that the stability of the genome must be under continuous surveillance. This is accomplished by DNA repair mechanisms, which have evolved to remove or to tolerate pre-cytotoxic, pre-mutagenic and pre-clastogenic DNA lesions in an error-free, or in some cases, error-prone way. Defects in DNA repair give rise to hypersensitivity to DNA-damaging agents, accumulation of mutations in the genome and finally to the development of cancer and various metabolic disorders. The importance of DNA repair is illustrated by DNA repair deficiency and genomic instability syndromes, which are characterised by increased cancer incidence and multiple metabolic alterations. Up to 130 genes have been identified in humans that are associated with DNA repair. This review is aimed at updating our current knowledge of the various repair pathways by providing an overview of DNA-repair genes and the corresponding proteins, participating either directly in DNA repair, or in checkpoint control and signaling of DNA damage.

Apoptosis in malignant glioma cells triggered by the temozolomide-induced DNA lesion O6-methylguanine

Methylating drugs such as temozolomide (TMZ) are widely used in the treatment of brain tumours (malignant gliomas). The mechanism of TMZ-induced glioma cell death is unknown. Here, we show that malignant glioma cells undergo apoptosis following treatment with the methylating agents N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and TMZ. Cell death determined by colony formation and apoptosis following methylation is greatly stimulated by p53. Transfection experiments with O6-methylguanine-DNA methyltransferase (MGMT) and depletion of MGMT by O6-benzylguanine showed that, in gliomas, the apoptotic signal originates from O6-methylguanine (O6MeG) and that repair of O6MeG by MGMT prevents apoptosis. We further demonstrate that O6MeG-triggered apoptosis requires Fas/CD95/Apo-1 receptor activation in p53 non-mutated glioma cells, whereas in p53 mutated gliomas the same DNA lesion triggers the mitochondrial apoptotic pathway. This occurs less effectively via Bcl-2 degradation and caspase-9, -2, -7 and -3 activation. O6MeG-triggered apoptosis in gliomas is a late response (occurring >120 h after treatment) that requires extensive cell proliferation. Stimulation of cell cycle progression by the Pasteurella multocida toxin promoted apoptosis whereas serum starvation attenuated it. O6MeG-induced apoptosis in glioma cells was preceded by the formation of DNA double-strand breaks (DSBs), as measured by γH2AX formation. Glioma cells mutated in DNA-PKcs, which is involved in non-homologous end-joining, were more sensitive to TMZ-induced apoptosis, supporting the involvement of DSBs as a downstream apoptosis triggering lesion. Overall, the data demonstrate that cell death induced by TMZ in gliomas is due to apoptosis and that determinants of sensitivity of gliomas to TMZ are MGMT, p53, proliferation rate and DSB repair.

DNA damage-induced cell death: from specific DNA lesions to the DNA damage response and apoptosis.

DNA damaging agents are potent inducers of cell death triggered by apoptosis. Since these agents induce a plethora of different DNA lesions, it is firstly important to identify the specific lesions responsible for initiating apoptosis before the apoptotic executing pathways can be elucidated. Here, we describe specific DNA lesions that have been identified as apoptosis triggers, their repair and the signaling provoked by them. We discuss methylating agents such as temozolomide, ionizing radiation and cisplatin, all of them are important in cancer therapy. We show that the potentially lethal events for the cell are O(6)-methylguanine adducts that are converted by mismatch repair into DNA double-strand breaks (DSBs), non-repaired N-methylpurines and abasic sites as well as bulky adducts that block DNA replication leading to DSBs that are also directly induced following ionizing radiation. Transcriptional inhibition may also contribute to apoptosis. Cells are equipped with sensors that detect DNA damage and relay the signal via kinases to executors, who on their turn evoke a process that inhibits cell cycle progression and provokes DNA repair or, if this fails, activate the receptor and/or mitochondrial apoptotic cascade. The main DNA damage recognition factors MRN and the PI3 kinases ATM, ATR and DNA-PK, which phosphorylate a multitude of proteins and thus induce the DNA damage response (DDR), will be discussed as well as the downstream players p53, NF-κB, Akt and survivin. We review data and models describing the signaling from DNA damage to the apoptosis executing machinery and discuss the complex interplay between cell survival and death.

O6-methylguanine DNA methyltransferase and p53 status predict temozolomide sensitivity in human malignant glioma cells

Temozolomide (TMZ) is a methylating agent which prolongs survival when administered during and after radiotherapy in the first‐line treatment of glioblastoma and which also has significant activity in recurrent disease. O6‐methylguanine DNA methyltransferase (MGMT) is a DNA repair enzyme attributed a role in cancer cell resistance to O6‐alkylating agent‐based chemotherapy. Using a panel of 12 human glioma cell lines, we here defined the sensitivity to TMZ in acute cytotoxicity and clonogenic survival assays in relation to MGMT, mismatch repair and p53 status and its modulation by dexamethasone, irradiation and BCL‐XL. We found that the levels of MGMT expression were a major predictor of TMZ sensitivity in human glioma cells. MGMT activity and clonogenic survival after TMZ exposure are highly correlated (p < 0.0001, r2 = 0.92). In contrast, clonogenic survival after TMZ exposure does not correlate with the expression levels of the mismatch repair proteins mutS homologue 2, mutS homologue 6 or post‐meiotic segregation increased 2. The MGMT inhibitor O6‐benzylguanine sensitizes MGMT‐positive glioma cells to TMZ whereas MGMT gene transfer into MGMT‐negative cells confers protection. The antiapoptotic BCL‐XL protein attenuates TMZ cytotoxicity in MGMT‐negative LNT‐229 but not in MGMT‐positive LN‐18 cells. Neither ionizing radiation (4 Gy) nor clinically relevant concentrations of dexamethasone modulate MGMT activity or TMZ sensitivity. Abrogation of p53 wild‐type function strongly attenuates TMZ cytotoxicity. Conversely, p53 mimetic agents designed to stabilize the wild‐type conformation of p53 sensitize glioma cells for TMZ cytotoxicity. Collectively, these results suggest that the determination of MGMT expression and p53 status will help to identify glioma patients who will or will not respond to TMZ.

DNA damage-triggered apoptosis: critical role of DNA repair, double-strand breaks, cell proliferation and signaling

Genotoxic DNA damaging agents may activate both membrane death receptors and the endogenous mitochondrial damage pathway leading to cell death via apoptosis. Here, apoptotic responses in cells exhibiting a defect in various DNA repair pathways such as alkyltransferase, base excision repair, nucleotide excision repair and mismatch repair are reviewed. The HSVTk/ganciclovir and VZV/BVDU suicide system will also be discussed. Data are available to show that critical DNA damage triggers apoptosis in a DNA replication dependent way by activating the mitochondrial damage pathway in fibroblasts. It is proposed that DNA double-strand breaks (DSBs) are common ultimate apoptosis-triggering lesions arising from primary DNA lesions during DNA replication. Thus, DNA replication is a necessary component in DNA damage-triggered apoptosis, at least in fibroblasts treated with genotoxins not inducing DSBs themselves. For methylating agents inducing O(6)-methylguanine, an additional requirement is mismatch repair provoking DSB formation that triggers Bcl-2 decline and caspase-9/-3 activation. This occurs independent of p53 since most of the repair deficient cell lines under study were mutated for p53. Moreover, p53 knockout fibroblasts are more sensitive to methylating agents and UV light than p53 wt cells, suggesting p53 to play a protective rather than a pro-apoptotic role in this cell system, probably by its involvement in DNA repair. However, for lymphoblastoid cells p53 wt variants are more sensitive to DNA damage indicating that p53 participates in apoptotic signaling in a cell type-specific fashion. The role of topoisomerase II inhibitors and c-Fos/AP-1 in apoptosis will also be discussed.

Cisplatin resistance: preclinical findings and clinical implications.

Cisplatin is used for the treatment of many types of solid cancers. While testicular cancers respond remarkably well to cisplatin, the therapeutic efficacy of cisplatin for other solid cancers is limited because of intrinsic or acquired drug resistance. Our understanding about the mechanisms underlying cisplatin resistance has largely arisen from studies carried out with cancer cell lines in vitro. The process of cisplatin resistance appears to be multifactorial and includes changes in drug transport leading to decreased drug accumulation, increased drug detoxification, changes in DNA repair and damage bypass and/or alterations in the apoptotic cell death pathways. Translation of these preclinical findings to the clinic is emerging, but still scarce. The present review describes and discusses the clinical relevance of in vitro models by comparing the preclinical findings to data obtained in clinical studies.

DNA damage and the balance between survival and death in cancer biology

DNA is vulnerable to damage resulting from endogenous metabolites, environmental and dietary carcinogens, some anti-inflammatory drugs, and genotoxic cancer therapeutics. Cells respond to DNA damage by activating complex signalling networks that decide cell fate, promoting not only DNA repair and survival but also cell death. The decision between cell survival and death following DNA damage rests on factors that are involved in DNA damage recognition, and DNA repair and damage tolerance, as well as on factors involved in the activation of apoptosis, necrosis, autophagy and senescence. The pathways that dictate cell fate are entwined and have key roles in cancer initiation and progression. Furthermore, they determine the outcome of cancer therapy with genotoxic drugs. Understanding the molecular basis of these pathways is important not only for gaining insight into carcinogenesis, but also in promoting successful cancer therapy. In this Review, we describe key decision-making nodes in the complex interplay between cell survival and death following DNA damage.

Rho GTPases: Promising Cellular Targets for Novel Anticancer Drugs

Ras-homologous (Rho) GTPases play a pivotal role in the regulation of numerous cellular functions associated with malignant transformation and metastasis. Rho GTPases are localized at membranes and become activated upon stimulation of cell surface receptors. In their GTP-bound (=active) state, Rho proteins bind to effector proteins, thereby triggering specific cellular responses. Members of the Rho family of small GTPases are key regulators of actin reorganization, cell motility, cell-cell and cell-extracellular matrix (ECM) adhesion as well as of cell cycle progression, gene expression and apoptosis. Each of these functions is of importance for the development and progression of cancer. Furthermore, Rho guanine exchange factors (GEFs) are often oncogenic and the expression level of Rho GTPases frequently increases with malignancy. Rho proteins also affect cellular susceptibility to DNA damaging agents, including antineoplastic drugs and ionizing radiation (IR). Thus, modulation of Rho driven mechanisms may influence the therapeutic efficiency and/or the side effects of conventional antineoplastic therapy. Because of their pleiotropic functions, Rho proteins appear to be promising targets for the development of novel anticancer drugs. Experimental approaches to inhibit Rho (and Ras) have focused on the attenuation of their C-terminal isoprenylation. This is because C-terminal lipid modification is required for correct intracellular localization and function of Rho/Ras. Inhibitors of farnesyltransferase (FTI), geranylgeranyltransferase (GGTI) as well as of HMG-CoA-reductase (i. e. statins) have been investigated with respect to their usefulness in tumor therapy. The studies showed that these compounds affect tumor progression and furthermore have impact on the frequency of cell death induced by tumor therapeutics. A possible drawback of inhibitors of isoprenylation is their poor selectivity for individual Rho GTPases. Therefore, specific inhibitors of individual Rho functions (notably RhoA-, RhoB-, Rac1- or Cdc42-related functions) are predicted to be of great therapeutic benefit. Indeed, compounds developed as specific inhibitors of the RhoA-effector molecule Rho-kinase (ROK) have been demonstrated to exert anti-metastatic activity in vivo.

Regulation of Repair of Alkylation Damage in Mammalian Genomes

Publisher Summary This chapter discusses the current understanding of the molecular basis of the regulation of alkylation damage repair in mammals. Such molecular studies were not possible until the recent success in the cloning of the alkylation repair genes. This chapter first explains the basic mechanisms of repair proteins. The availability of nucleic-acid and antibody probes for many of the alkylation repair genes and proteins, and elucidation of the structure and identification of the regulatory elements of these genes, provide an opportunity for a comprehensive understanding of regulation of alkylation damage repair. The prospect of large-scale production of the human alkylation repair proteins in E . coli for the subsequent determination of their structure by X-ray crystallography and NMR looks quite good. In contrast to inhibition of repair genes at the level of their expression, these genes could also be inactivated in cultured cells by homologous recombination. Starting with mutations in repair genes of pluripotent embryonic stem cells, repair-deficient or repair-negative mice could be generated. Such animals may make excellent models for mutagen, carcinogen, and aging studies.