Tamara T. Lah

Spontaneous Malignant Transformation of Human Mesenchymal Stem Cells Reflects Cross-Contamination: Putting the Research Field on Track - Letter

Several groups, including ours, have published results showing spontaneous transformation of human mesenchymal stem cells (MSC). Recently, we reported in this journal spontaneous transformation of bone marrow-derived human MSC (hMSC), isolated and expanded independently in two laboratories ([1][1

Selective suppression of cathepsin L by antisense cDNA impairs human brain tumor cell invasion in vitro and promotes apoptosis

Invasion and metastasis of certain tumors are accompanied by increased mRNA protein levels and enzymatic activity of cathepsin L. Cathepsin L has also been suggested to play a role in the proteolytic cascades associated with apoptosis. To investigate the role of cathepsin L in brain tumor invasion and apoptosis, the human glioma cell line, IPTP, was stably transfected with full-length antisense and sense cDNA of cathepsin L. Down-regulation of cathepsin L by antisense cDNA significantly impaired (up to 70%) glioma cell invasion in vitro and markedly increased glioma cell apoptosis induced by staurosporine. Compared to control and parental cell lines, antisense down-regulation of cathepsin L was associated with an earlier induction of caspase-3 activity. Up-regulation of cathepsin L activity by sense cDNA was associated with reduced apoptosis and later induction of caspase-3 activity. Moreover, down-regulation of cathepsin L lowered the expression of antiapoptotic protein Bcl-2, whereas up-regulation increased the expression of Bcl-2, indicating that cathepsin L acts upstream of caspase-3. These data show that cathepsin L is an important protein mediating the malignancy of gliomas and its inhibition may diminish their invasion and lead to increased tumor cell apoptosis by reducing apoptotic threshold.

Patterns of microcystin-LR induced alteration of the expression of genes involved in response to DNA damage and apoptosis☆

Microcystins (MCs) are hepatotoxic cyclic heptapeptides produced by freshwater cyanobacteria. They are inhibitors of serine/threonine protein phosphatases 1A and 2A and are involved in liver tumour promotion. Several recent studies indicated that MCs are genotoxic and may also act as tumour initiators. Based on our previous results showing that microcystin-LR (MCLR) induces DNA damage in HepG2 cells, we have now explored the effect of MCLR on the expression of selected genes known to be involved in the cell response to DNA damage and apoptosis. The HepG2 cells were exposed to non-cytotoxic concentrations (0.01, 0.1 and 1 microg/ml) of MCLR for various periods of time (2-16 h) and the mRNA expression was determined with the quantitative real-time polymerase chain reaction (QRT-PCR). We found a significantly elevated expression of tumour suppressor gene p53 and its downstream-regulated genes involved in DNA repair and cell cycle regulation (p21, gadd 45a, mdm2), as well as increased expression of the pro-apoptotic gene bax, but no alterations of the anti-apoptotic bcl-2. Up-regulation of the expression of mdm2, p21 and gadd45a provides strong support for our previous suggestion that MCLR is a genotoxic carcinogen. The increased ratio of expression of bax to that of bcl-2 induced by MCLR suggests that apoptosis in HepG2 cells proceeds via the mitochondrial pathway.

Cadmium inhibits repair of UV-, methyl methanesulfonate- and N-methyl-N-nitrosourea-induced DNA damage in Chinese hamster ovary cells.

The co-genotoxic effects of cadmium are well recognized and it is assumed that most of these effects are due to the inhibition of DNA repair. We used the comet assay to analyze the effect of low, non-toxic concentrations of CdCl2 on DNA damage and repair-induced in Chinese hamster ovary (CHO) cells by UV-radiation, by methyl methanesulfonate (MMS) and by N-methyl-N-nitrosourea (MNU). The UV-induced DNA lesions revealed by the comet assay are single-strand breaks which are the intermediates formed during nucleotide excision repair (NER). In cells exposed to UV-irradiation alone the formation of DNA strand breaks was rapid, followed by a fast rejoining phase during the first 60 min after irradiation. In UV-irradiated cells pre-exposed to CdCl2, the formation of DNA strand breaks was significantly slower, indicating that cadmium inhibited DNA damage recognition and/or excision. Methyl methanesulfonate and N-methyl-N-nitrosourea directly alkylate nitrogen and oxygen atoms of DNA bases. The lesions revealed by the comet assay are mainly breaks at apurinic/apyrimidinic (AP) sites and breaks formed as intermediates during base excision repair (BER). In MMS treated cells the initial level of DNA strand breaks did not change during the first hour of recovery; thereafter repair was detected. In cells pre-exposed to CdCl2 the MMS-induced DNA strand breaks accumulated during the first 2h of recovery, indicating that AP sites and/or DNA strand breaks were formed but that further steps of BER were blocked. In MNU treated cells the maximal level of DNA strand breaks was detected immediately after the treatment and the breaks were repaired rapidly. In CdCl2 pre-treated cells the formation of MNU-induced DNA single-strand breaks was not affected, while the repair was slower, indicating inhibition of polymerization and/or the ligation step of BER. Cadmium thus affects the repair of UV-, MMS- and MNU-induced DNA damage, providing further evidence, that inhibition of DNA repair is an important mechanism of cadmium induced mutagenicity and carcinogenicity.

Antiprotease therapy in cancer: hot or not?

The activity of a set of peptidases (proteases) involved in cancer progression is collectively known as the cancer ‘degradome’. Invasion and metastasis were initially considered as late events in cancer development and the processes in which proteases were involved. However, recent studies indicate that invasion and metastasis are not late events, but can occur during early stages as well. Moreover, other processes occurring in various stages of cancer progression are also protease-dependent, such as (upregulation of) cell proliferation, (downregulation of) apoptosis, involvement of white blood cells, angiogenesis and induction of multi-drug resistance. Proteolytic activity in tumours is regulated in a complex manner, as both genetically unstable cancer cells and stable stromal cells, such as fibroblasts, endothelial cells and inflammatory cells, are involved. In vitro studies and studies using animal models have clearly shown protease dependency of many processes in carcinogenesis. However, clinical trials using protease inhibitors have thus far been unsuccessful except for a few applications of matrix metalloprotease (MMP) inhibitors when used in combination with cytostatic anticancer agents and/or in the early stages of cancer. Antithrombotics, such as low-molecular-weight heparin and warfarin, were also successful in clinical trials, probably by interfering with proteases of the coagulation cascade. The two-way association between cancer and thrombosis has long been recognised in the clinic. The poor outcome of other clinical trials of protease inhibitors is probably due to the late stages of cancer of the patient populations included, and the limited understanding of the complex regulation and effects of the activity of the various proteases in tumours depending on, among others, tumour type and stage, interactions between the cancer cells, other cells and the extracellular matrix in tumours. Therefore, a better fundamental understanding of the proteolytic complexity in tumours is essential before clinical trials can be rationally designed. At present, antithrombotics, the urokinase-type plasminogen activator system, the membrane-bound membrane-type 1-MMP, cathepsin L and the proteasome seem the most promising candidates as targets for anticancer strategies in early stages of cancer in combination with cytotoxic drugs. Moreover, metronomic therapy is an attractive approach using low doses of inhibitors for prolonged periods of time without interruption to specifically target endothelial cells that are involved in angiogenesis.

Lysosomal Enzymes, Cathepsins in Brain Tumour Invasion

The expression patterns of different classes of peptidases in central nervous system (CNS) tumours have been most extensively studied in astrocytomas and meningiomas. Although the two types of tumours are very different in most respects, both may invade locally into normal brain. This process of invasion includes increased synthesis and secretion of lysosomal proteolytic enzymes – cathepsins.Aspartic endopeptidase cathepsin (Cat) D levels were found to be elevated in high-grade astrocytoma and partial inhibition of glioblastoma cell invasion by anti-Cat D antibody suggests that the enzyme activity is involved in the invasion process. Several studies on cysteine endopeptidase (CP) Cat B in gliomas agreed that transcript abundance, protein level and activity of Cat B increased in high-grade astrocytoma cultures compared with low-grade astrocytoma cultures and normal brain. Moreover, in glioma biopsies Cat B levels correlated with evidence of clinical invasion and it has been demonstrated that Cat B both in tumour cells and in endothelial cells can serve as a new biological marker for prognosis in glioblastoma patients. A high level of Cat B protein was also a diagnostic marker for invasive types of meningioma, distinguishing between histomorphologically benign, but invasive meningiomas and noninvasive, so-called clear–benign meningiomas. Cat L was also significantly increased in high-grade astrocytoma compared with low-grade astrocytoma and normal brain. Specific Cat L antibodies and antisense Cat L RNA transfection significantly lowered glioblastoma cell invasion. In meningioma, Cat L was a less-significant marker of invasion than Cat B. In contrast to cathepsins, the activities of endogenous cysteine peptidase inhibitors (CPIs), including stefins, cystatins and kininogens, were significantly higher in benign and atypical meningioma cell extracts than in malignant meningioma, and low-grade compared to high-grade astrocytoma. However, very low levels of stefins A and B were found in meningioma and glioblastoma tissues. Further studies on the expression levels and balance between cysteine endopeptidases (CPs) and CPIs would improve the clinical application of cathepsins in prognosis, which would lead to more-informed therapeutic strategies.

Invasiveness of transformed human breast epithelial cell lines is related to cathepsin B and inhibited by cysteine proteinase inhibitors.

Abstract The activities of the lysosomal cysteine proteinases cathepsin B and L are regulated by their endogenous inhibitors, stefins A and B, and cystatin C, and their imbalance may be associated with increased invasiveness and development of the malignant cell phenotype. The aim of this study was to investigate mRNA, protein and activity levels of the above proteins in relation to in vitro invasiveness and to the reported in vivo tumorigenicity of four human breast tumor cell lines: the spontaneously immortalized cell line MCF10A, its cHaras transfectant MCF10AT, and two tumorigenic derivative cell lines, MCF10AT-Ca1a and MCF10AT-Ca1d. Invasiveness did not correlate with tumorigenicity, since the MCF10AT cell was the most invasive and the remaining three were at about half of its level. Cathepsin B expression paralleled the in vitro invasiveness through matrigel at all levels of expression, but cathepsin L did not. Stefin levels were elevated severalfold in the tumorigenic cell lines, but not in MCF10AT. The hypothesis that cathepsin B plays an active role in the invasion of breast cancer cell lines was confirmed by the fact that synthetic cysteine proteinase inhibitors, particularly those selective for cathepsin B, significantly reduced the invasion of the MCF10AT cells.

Effects of model organophosphorous pesticides on DNA damage and proliferation of HepG2 cells.

Organophosphorous compounds (OPs) are commonly used pesticides. The primary mechanism of OP toxicity is the inhibition of acetylcholine esterase in the nervous system leading to a variety of acute and chronic effects. Recent studies have revealed several other targets of OPs that disturb noncholinergic biological systems. We investigated whether low concentrations of model OPs—methyl parathion (PT), methyl paraoxon (PO), and dimefox (DF)—induce DNA damage and/or affect cell proliferation in human hepatoma HepG2 cells. Genotoxicity of OPs was evaluated using the comet assay. The effect on cell proliferation was tested using the MTT assay and proliferation marker Ki‐67 immunocytochemistry. The effects of OPs on mRNA expression of the DNA damage responsivegenes p53, p21, GADD45α, and MDM2 were determined using qRT‐PCR. PT induced DNA damage at lower concentrations (1 μg/mL) than PO (100 μg/mL), whereas DF did not induce DNA damage. PT and PO caused a reduction of cell proliferation at their highest concentrations (100 μg/mL), while DF increased cell proliferation at all concentrations used (0.01–100 μg/mL). PT and PO upregulated expression of DNA damage responsive genes, while DF upregulated expression of p53, downregulated expression of p21, and had no effect on the expression of MDM2 and GADD45α. We conclude that PT and PO are genotoxic, while DF shows mitogenic activity. An important finding of this study is that PT had higher genotoxic potential than PO, which warrants for further investigations to correctly evaluate the hazards of exposure to these chemicals. Environ. Mol. Mutagen., 2008.

Cathepsins B and L are markers for clinically invasive types of meningiomas.

OBJECTIVEMeningiomas are benign neoplasms that derive from coverings of the brain. Approximately 10% of benign tumors progress into atypical, malignant tumors, thus constituting a subset of histopathologically benign tumors that are clinically invasive. The aim of this study was to evaluate cathepsins B and L and their inhibitors as new prognostic factors that could distinguish malignant from benign forms of meningiomas. METHODSUsing immunohistochemical analysis and specific monoclonal antibodies, we evaluated the levels of cathepsins B and L and the levels of the endogenous cysteine proteinase inhibitors stefin A and cystatin C in 88 meningiomas. Immunohistochemical scores were determined as the sum of the frequency (0–3) and intensity (0–3) of immunolabeling of the tumor cells. RESULTSOf the 88 tumors studied, 67 were benign meningiomas and 21 were atypical meningiomas. Among the benign group, nine tumors had certain features of malignancy. These tumors were classified as border benign meningiomas, and the rest were classified as clear benign meningiomas. A high immunohistochemical score (4–6) for cathepsin B was more frequent in atypical tumors than in clear benign tumors (P < 0.001). Compared with clear benign tumors, higher cathepsin B immunohistochemical scores were found in atypical tumors (P < 0.001) and border benign tumors (P < 0.03). No statistical difference in immunohistochemical staining of cathepsin B was found between atypical meningiomas and border benign meningiomas. Higher expression of cathepsin L was found in atypical tumors as compared with clear benign tumors (P < 0.03), but it was not observed in border benign as compared with clear benign meningiomas. No immunostaining for stefin A and cystatin C was detected in any of the tumors. CONCLUSIONWe show that the levels of cathepsin B and cathepsin L antigens are significantly higher in invasive types of benign meningioma. Specifically, cathepsin B may be used as a diagnostic marker to distinguish histomorphologically benign but invasive meningiomas from histomorphologically clear benign tumors.

Glioblastoma and endothelial cells cross-talk, mediated by SDF-1, enhances tumour invasion and endothelial proliferation by increasing expression of cathepsins B, S, and MMP-9.

Malignant glioma is characterized by rapid proliferation, high invasiveness into the surrounding brain and increased vascularity. The aim of the study was to explain the observation that glioblastoma invasion often occurs along existing vasculature, suggesting interactions between the two types of cells. Using the in vitro model, we demonstrate that co-culturing of U87 (human glioblastoma) cells with HMEC-1 (human microvascular endothelial) cells increases the invasiveness of the U87 cells. The enhanced invasiveness correlates with increased expression of MMP-9 in both U87 and HMEC-1 cells, increased expression of cysteine cathepsins B and S and down-regulation of endogenous cell adhesion molecule NCAM in U87 cells. On the other hand, U87 tumour cells significantly enhance the proliferation of co-cultured endothelial cells by a mechanism involving cathepsin B, but not cathepsin S. Furthermore, we demonstrated that increased cell expression and activity of MMP-9 in cell microenvironment is mediated via secretion of SDF-1 by HMEC-1 cells. Selective SDF-1 inhibition impaired the enhanced U87 cell invasion, mostly via down-regulation of MMP-9, but did not alter cathepsin B, although the latter is more relevant for the invasion of U87 cells in mono-culture. Taken together, our study suggests that glioblastoma cells may be attracted by endothelial cells, enhancing their proliferation and underlines the importance of SDF-1, cathepsin B and MMP-9 in the cross-talk between these cells in normoxic conditions. This notion contributes to better understanding and suggests further investigations of the paracrine mechanisms, regulating glioma angiogenesis.