Introduction: Oral tongue squamous cell carcinoma (OTSCC) is the most common cancer of the oral c... more Introduction: Oral tongue squamous cell carcinoma (OTSCC) is the most common cancer of the oral cavity. Contradictory results have been observed on the involvement of herpes simplex virus 1 (HSV-1) in oral squamous cell carcinomas. Here, we aimed to study the predominance of HSV-1 or HSV-2 in oral HSV infections and to investigate the presence of HSV-1 in OTSCC and its effect on carcinoma cell viability and invasion.Methods: The distribution of HSV types one and two in diagnostic samples taken from suspected oral HSV infections was determined from the Helsinki University Hospital Laboratory database. We then analysed 67 OTSCC samples for HSV-1 infection using immunohistochemical staining. We further tested the effects of HSV-1 using six concentrations (0.00001–1.0 multiplicity of infection [MOI]) on viability and two concentrations (0.001 and 0.1 MOI) on invasion of highly invasive metastatic HSC-3 and less invasive primary SCC-25 OTSCC cell lines using MTT and Myogel-coated Transwe...
ObjectiveAlthough there have been remarkable achievements in the molecular landscape of oral squa... more ObjectiveAlthough there have been remarkable achievements in the molecular landscape of oral squamous cell carcinoma (OSCC) in recent years, bringing advances in the understanding of its pathogenesis, development and progression, little has been applied in the prognosis and choosing the optimal treatment. In this study, we explored the influence of the stress induced phosphoprotein 1 (STIP1), which is frequently reported to be highly expressed in many cancers, in OSCCs.MethodsSTIP1 expression was assessed in the TCGA database and in two independent cohorts by immunohistochemistry. Knockdown strategy was applied in OSCC cell lines to determine the impact of STIP1 on viability, proliferation, migration and invasion. The zebrafish model was applied for studying tumor formation and metastasis in vivo. The association of STIP1 and miR-218-5p was explored by bioinformatics and mimics transfection.ResultsSTIP1 was highly expressed in OSCCs and significantly associated with shortened surviv...
Proper functioning of each secretory and endocytic compartment relies on its unique pH micro-envi... more Proper functioning of each secretory and endocytic compartment relies on its unique pH micro-environment that is known to be dictated by the rates of V-ATPase-mediated H+ pumping and its leakage back to the cytoplasm via an elusive “H+ leak” pathway. Here, we show that this proton leak across Golgi membranes involves AE2a (SLC4A2a)-mediated bicarbonate-chloride exchange, as it is strictly dependent on both bicarbonate import (in exchange of chloride export) and the AE2a expression level in the cells. Imported bicarbonate anions and luminal protons then facilitate a common buffering reaction that yields carbon dioxide and water before their egress back to the cytoplasm via diffusion or water channels. The high surface-volume ratio of flattened Golgi cisternae helps this process, as their shape is optimal for water and gas exchange. Interestingly, this pathway is often upregulated in cancers and established cancer cell lines, and responsible for their markedly elevated Golgi resting p...
Conventional chemotherapeutic agents are nonselective, often resulting in severe side effects and... more Conventional chemotherapeutic agents are nonselective, often resulting in severe side effects and the development of resistance. Therefore, new molecular-targeted therapies are urgently needed to be integrated into existing treatment regimens. Here, we performed a high-throughput compound screen to identify a synergistic interaction between ionizing radiation and 396 anticancer compounds. The assay was run using five human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) cell lines cultured on the human tumor-derived matrix Myogel. Our screen identified several compounds with strong synergistic and antagonistic effects, which we further investigated using multiple irradiation doses. Navitoclax, which emerged as the most promising radiosensitizer, exhibited synergy with irradiation regardless of the p53 mutation status in all 13 HNSCC cell lines. We performed a live cell apoptosis assay for two representative HNSCC cell lines to examine the effects of navit...
Cancer invasiveness and metastatic spread are the main causes of cancer-associated deaths, yet su... more Cancer invasiveness and metastatic spread are the main causes of cancer-associated deaths, yet successful therapies for their prevention are currently non-existing. Here we exploit cancer cell reprogramming, a strategy developed earlier to prevent tumour growth, to show that cancer cell invasiveness can be reversed via restoring their Golgi pH homeostasis and normal glycosylation status. Specifically, we demonstrate that AE2a (SLC4A2a)-mediated bicarbonate-chloride exchange controls proton leakage across Golgi membranes via a well-known buffering reaction that produces water and carbon dioxide from luminal protons and bicarbonate anions. Its forced overexpression in non-malignant cells, or upregulation in certain cancer cells in vitro and in vivo, also coincides with elevated Golgi resting pH, the synthesis of invasion and metastasis-promoting glycans and a shorter life span of cancer patients. Finally, AE2 knockdown in cancer cells blocked their invasiveness via restoring their Gol...
Abstract Lysyl hydroxylase (LH) catalyzes the post-translational formation of hydroxylysines in c... more Abstract Lysyl hydroxylase (LH) catalyzes the post-translational formation of hydroxylysines in collagens and collagenous proteins. Three lysyl hydroxylase isoforms, LH1, LH2 and LH3, have been identified from different species. In addition, LH2 has two alternatively spliced forms, LH2a and LH2b. The hydroxylysines have an important role in the formation of the intermolecular collagen crosslinks that stabilize the collagen fibrils. Some of the hydroxylysine residues are further glycosylated. In this thesis the substrate amino acid sequence specificities of the LH isoforms were analyzed using synthetic peptide substrates. The data did not indicate strict amino acid sequence specificity for the LH isoforms. However, there seemed to be a preference for some sequences to be bound and hydroxylated by a certain isoform. Galactosylhydroxylysyl glucosyltransferase (GGT) catalyzes the formation of glucosylgalactosylhydroxylysine. In this study, LH3 was shown to be a multifunctional enzyme, possessing LH and GGT activities. The DXD-like motif, characteristic of many glycosyltransferase families, and the conserved cysteine and leucine residues in the N-terminal part of the LH3 molecule were critical for the GGT activity, but not for the LH activity of the molecule. The GGT/LH3 protein level was found to be decreased in skin fibroblasts and in the culture media of cells collected from members of a Finnish epidermolysis bullosa simplex (EBS) family, which was earlier reported to have a deficiency of GGT activity. In this study, we showed that the reduction of enzyme activity is not due to a mutation or lower expression of the LH3 gene. Our data indicate that the decreased GGT/LH3 activity in cells has an effect on the deposition and organization of the key extracellular matrix components, collagen types VI and I and fibronectin, and these changes are transmitted to the cytoskeletal network. These findings underline LH3 as an important extracellular regulator
Oral carcinoma is one of the most aggressive cancers, and despite the advances in the therapy, it... more Oral carcinoma is one of the most aggressive cancers, and despite the advances in the therapy, its mortality is still high. An attention in cancer treatment has focused on natural compounds due to their potential beneficial effects on human health. In this study, the effects of dietary supplement Fermented Wheat Germ Extract (FWGE) on oral tongue squamous cell carcinoma (OTSCC) cells were investigated in vitro using three cell lines (HSC-3, SAS, SCC-25) with variable aggressiveness. The cell viability was significantly decreased by the treatment with high concentration of FWGE in every cell line. Regarding migration and invasion, HSC-3 and SCC-25 cells were most sensitive to FWGE since their movement was significantly reduced with 5 and 10 mg/ml FWGE, while SAS was inhibited only with 10 mg/ml FWGE. Chemotherapeutic compounds (cisplatin and 5-fluorouracil) significantly reduced all OTSCC cells viability. Importantly, combination of these drugs with 10 mg/ml FWGE significantly decreased the cell viability compared to the treatment with the chemotherapeutics or FWGE alone. Based on these in vitro experiments, the use of FWGE seems to improve the anticancer effects on OTSCC cells. Further in vivo and clinical studies should be conducted to verify the positive effects of FWGE for OTSCC patients.
Proper functioning of each secretory and endocytic compartment relies on its unique pH micro-envi... more Proper functioning of each secretory and endocytic compartment relies on its unique pH micro-environment that is known to be dictated by the rates of V-ATPase-mediated H+ pumping and its leakage back to the cytoplasm via an elusive “H+ leak” pathway. Here, we show that this proton leak across Golgi membranes is mediated by the AE2a (SLC4A2a)-mediated bicarbonate-chloride exchange, as it is strictly dependent on bicarbonate import (in exchange for chloride export) and the expression level of the Golgi-localized AE2a anion exchanger. In the acidic Golgi lumen, imported bicarbonate anions and protons then facilitate a common buffering reaction that yields carbon dioxide and water before their egress back to the cytoplasm via diffusion or water channels. The flattened morphology of the Golgi cisternae helps this process, as their high surface-volume ratio is optimal for water and gas exchange. Interestingly, this net acid efflux pathway is often upregulated in cancers and established ca...
Oral tongue squamous cell carcinoma (OTSCC), the most common cancer in the oral cavity, is aggres... more Oral tongue squamous cell carcinoma (OTSCC), the most common cancer in the oral cavity, is aggressive and its incidence is increasing globally. Human host defense cationic antimicrobial peptide-18/antimicrobial peptide leucine-leucine-37 (hCAP18/LL-37) plays a complex role in various types of cancers. In the present study, we characterized the effects of exogenous LL-37 on three OTSCC cell lines and determined the expression of hCAP18/LL-37 in oral dysplastic and OTSCC patient samples. Our data revealed that LL-37, especially in high doses, mostly reduced the proliferation of OTSCC cells, but the effect was fluctuating. However, LL-37 stimulated the migration and invasion of OTSCC cells. The high dose of LL-37 also increased the amount of total epidermal growth factor receptor (EGFR) probably due to stabilization of the receptor to the plasma membrane. However, activation of EGFR downstream pathways was mostly decreased. Our immunohistochemical analysis showed that the hCAP18/LL-37 expression was higher in normal/mild dysplasia than in moderate/severe dysplasia and OTSCC. The hCAP18/LL-37 expression did not correlate with clinicopathological features or outcome of OTSCC patients. Our data suggest that LL-37 has a fluctuating effect on proliferation, migration and invasion of OTSCC cells, but it does not seem to play a role in the progression of OTSCC.
In vitro cancer drug testing carries a low predictive value. We developed the human leiomyoma–der... more In vitro cancer drug testing carries a low predictive value. We developed the human leiomyoma–derived matrix “Myogel” to better mimic the human tumor microenvironment (TME). We hypothesized that Myogel could provide an appropriate microenvironment for cancer cells, thereby allowing more in vivo–relevant drug testing. We screened 19 anticancer compounds, targeting the epidermal growth factor receptor (EGFR), MEK, and PI3K/mTOR on 12 head and neck squamous cell carcinoma (HNSCC) cell lines cultured on plastic, mouse sarcoma–derived Matrigel (MSDM), and Myogel. We applied a high-throughput drug screening assay under five different culturing conditions: cells in two-dimensional (2D) plastic wells and on top or embedded in Matrigel or Myogel. We then compared the efficacy of the anticancer compounds to the response rates of 19 HNSCC monotherapy clinical trials. Cancer cells on top of Myogel responded less to EGFR and MEK inhibitors compared to cells cultured on plastic or Matrigel. Howev...
Pro-tumorigenic activities of matrix metalloproteinase (MMP) 9 have been linked to many cancers, ... more Pro-tumorigenic activities of matrix metalloproteinase (MMP) 9 have been linked to many cancers, but recently the tumour-suppressing role of MMP9 has also been elucidated. The multifaceted evidence on this subject prompted us to examine the role of MMP9 in the behaviour of oral tongue squamous cell carcinoma (OTSCC) cells. We used gelatinase-specific inhibitor, CTT2, and short hairpin (sh) RNA gene silencing to study the effects of MMP9 on proliferation, motility and invasion of an aggressive OTSCC cell line, HSC-3. We found that the migration and invasion of HSC-3 cells were increased by CTT2 and shRNA silencing of MMP9. Proliferation, in turn, was decreased by MMP9 inhibition. Furthermore, arresten-overexpressing HSC-3 cells expressed increased levels of MMP9, but exhibited decreased motility compared with controls. Interestingly, these cells restored their migratory capabilities by CTT2 inhibition of MMP9. Hence, although higher MMP9 expression could give rise to an increased tumour growth in vivo due to increased proliferation, in some circumstances, it may participate in yet unidentified molecular mechanisms that reduce the cell movement in OTSCC. However, MMPs have far more complex roles in cancer, and some MMPs are now also shown to suppress some aspects of cancer progression (3). MMP9 has conventionally been considered as a pro-tumorigenic enzyme in oral squamous cell carcinoma (OSCC). A recent meta-analysis study showed that MMP9 overexpression is a predictor of poor prognosis in OTSCC patients (4). However, whether MMP9 expression in the most aggressive tumours enhances tumour progression or is more a consequence of tumour aggressiveness is unknown. Previous studies have revealed both pro-and anti-tumorigenic roles of MMP9 (reviewed in detail by Vilen et al. 2013), and its role in oral cancer is far from clear (5). Jordan et al. (2004) found that the mRNA levels of MMP9 were significantly higher in oral dysplasias that progressed to oral cancer than in those that did not (6). Pro-and active forms, total activities and the activation ratio of MMP9 were also significantly elevated in OSCC samples compared with their adjacent areas histologically rated as normal tissues (7). In contrast, Stokes et al. (2010) showed that MMP9 mRNA in primary tumours compared with adjacent peritumoral tissues was significantly decreased in head and neck squamous cell carcinomas with lymph node metastasis compared with non-metastatic tumours (8). Moreover, Lin et al. (2012) demonstrated that although patients with OSCC exhibit significantly higher levels of MMP9 than healthy controls, and even though MMP9 plasma levels are associated with more advanced clinical stages, MMP9 level was not associated with positive lymph node or distal metastasis (9). Evidence for the oncosuppressive role of MMP9 has also emerged for other cancers such as colitis-associated cancers (10) and breast cancer when MMP9 is produced by cancer cells (11). Recently, we have reported increased MMP9 expression in HSC-3 clone overexpressing MMP8 with decreased migration and invasion (12). Additionally, although anti-MMP2 and-9 peptides inhibit tongue carcinoma growth (13,14) and angiogenesis (13), they do not prevent the spread of carcinoma 4 cells in nude mice (14). Instead, when anti-MMP2 and-9 peptides were used in combination with proMMP9 targeting therapy, increased HSC-3 tumour growth was observed in mice (14). This study aims to elucidate the role of MMP9 in the behaviour of aggressive oral tongue carcinoma cells in vitro by using various genetically modified cell models with altered MMP9 expression as well as inhibition of MMP9 by gelatinase (MMP2 and-9) inhibitor peptide CTT2 (15). Materials and methods Cell Culture Native cell lines: Human tongue squamous cell carcinoma cell lines HSC-3 (Japanese Collection of Research Bioresources (JCRB) Cell Bank, JCRB0623), SAS (JCRB Cell Bank, JCRB0260), SCC-25 (American Type Culture Collection (ATCC), CRL 1628), human malignant melanoma G361 (ATCC, CRL-1424) and human Caucasian breast adenocarcinoma cell line MDA-MB-231 (ATCC, HTB-26) were cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12, 1:1 (Gibco) supplemented with 10% heat-inactivated foetal bovine serum (FBS, Gibco), 50 μg/ml ascorbic acid, 100 U/ml penicillin, 100 μg/ml streptomycin, 250 ng/ml amphotericin B and 0.4 µg/ml hydrocortisone (all from Sigma-Aldrich). Human embryonic kidney cell HEK-293 (ATCC, CRL-1573), human gingival fibroblasts (GFs) and human spontaneously immortalized keratinocytes (HaCaT) (16) were cultured in DMEM (Sigma-Aldrich) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml ascorbic acid, 250 ng/ml amphotericin B and 1 mM sodium pyruvate (all from Sigma-Aldrich). GFs used in this study were obtained from biopsies of healthy gingiva as described earlier (17). The human papillomavirus HPV16 immortalized human oral epithelial cells (IHGK) (18) were cultured in Keratinocyte-SFM (Gibco) supplemented with 5 ng/ml human recombinant epidermal growth factor, 50 µg/ml bovine pituitary extract (both from Gibco), 100 U/ml
Chemoradiation is an established approach in the treatment of advanced oral tongue squamous cell ... more Chemoradiation is an established approach in the treatment of advanced oral tongue squamous cell carcinoma (OTSCC), but therapy may cause severe side-effects due to signal interchanges between carcinoma and the tumour microenvironment (TME). In this study, we examined the potential use of our human 3D myoma disc and Myogel models in in vitro chemoradiation studies by analysing the effects of ionizing radiation (IR) and the combined effect of heparanase I (HPSE1) inhibitors and IR on OTSCC cell proliferation, invasion and MMP-2 and - 9 production. Finally, we analysed the long-term effects of IR by studying clones of previously irradiated and invaded HSC-3 cells. We found that in both human uterine leiomyoma-based extracellular matrix models IR inhibited the invasion of HSC-3 cells, but blocking HPSE1 activity combined with IR induced their invasion. Low doses of IR increased MMP expression and initiated epithelial-mesenchymal transition in cells cultured on myoma discs. We conclude ...
Sepsis delays wound re-epithelialization. In this study we explored the effect of human sepsis se... more Sepsis delays wound re-epithelialization. In this study we explored the effect of human sepsis sera as well as the effects of cytokines, growth factors and exosomes of sepsis sera treated normal fibroblasts (NF) on keratinocyte migration and proliferation in vitro. Serum samples were taken on days 1, 4, and 9 from 44 patients diagnosed with severe sepsis, and from 14 matching healthy controls. We evaluated the effects of sepsis serum with or without TNF-α, EGF, EGF receptor inhibitor or exosomes of sepsis sera treated NF on human keratinocyte (HaCaT) proliferation (BrdU assay), viability (MTT assay), and migration (horizontal wound healing model). Cytokine levels of sepsis and healthy sera were measured by multiplex assay. Comparisons between groups were carried out using SPSS statistics and P < 0.05 was considered significant. Severe-sepsis sera collected on days 1, 4, and 9 reduced keratinocyte proliferation by 6% (P = 0.005), 20% (P = 0.001), and 18% (P = 0.002), respectively,...
Post-translational modifications are necessary for collagen precursor molecules (procollagens) to... more Post-translational modifications are necessary for collagen precursor molecules (procollagens) to acquire final shape and function. However, the mechanism and contribution of collagen modifications that occur outside the endoplasmic reticulum and Golgi are not understood. We discovered that VIPAR, with its partner proteins, regulate sorting of lysyl hydroxylase 3 (LH3, also known as PLOD3) into newly identified post-Golgi collagen IV carriers and that VIPAR-dependent sorting is essential for modification of lysines in multiple collagen types. Identification of structural and functional collagen abnormalities in cells and tissues from patients and murine models of the autosomal recessive multisystem disorder Arthrogryposis, Renal dysfunction and Cholestasis syndrome caused by VIPAR and VPS33B deficiencies confirmed our findings. Thus, regulation of post-Golgi LH3 trafficking is essential for collagen homeostasis and for the development and function of multiple organs and tissues.
<p>(A) Amount of MBL-A was reduced in the serum of LH mutant mice (MUT) compared with the c... more <p>(A) Amount of MBL-A was reduced in the serum of LH mutant mice (MUT) compared with the corresponding wild type mice (WT). Amount of MBL-A was analyzed from the serum of the 2 months old male (n = 4 WT, 4 MUT) and female (n = 4 WT, 4 MUT) and 1 year old male (n = 4 WT, 3 MUT) and female (n = 5 WT, 6 MUT) by immunoblots. The values represent the average ± SD of the samples. MBL-A monomers in the serum of 2 months old female (B) and 1 year old male (C) wild type and LH mutant mice did not show significant differences in electrophoretic mobility. (D) The immunoblot analysis of liver homogenate (40 µg of soluble protein) of wild type and LH mutant male mice did not show differences in mobility of MBL-A monomers. (E) The amount of MBL-A in liver of LH mutant mice is higher than in wild type. Amount of MBL-A in liver was analyzed with immunoblots and α-tubulin was used as control to normalize the quantities of protein. In immunoblots B, C and D two representative wild type and LH samples are shown. MBL-A in LH mutant mice does not show doublet band, differing from the results of rat MBL-A overexpressed in cell culture system (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113498#pone-0113498-g001" target="_blank">Fig. 1A</a>). The values in figure A and E represent the average ± SD of the samples. P values were calculated using unpaired homoscedastic student t-test with two-tailed distribution. * p<0.05.</p
Lysyl hydroxylase 3 (LH3) is a multifunctional protein with lysyl hydroxylase, galactosyltransfer... more Lysyl hydroxylase 3 (LH3) is a multifunctional protein with lysyl hydroxylase, galactosyltransferase and glucosyltransferase activities. The LH3 has been shown to modify the lysine residues both in collagens and also in some collagenous proteins. In this study we show for the first time that LH3 is essential for catalyzing formation of the glucosylgalactosylhydroxylysines of mannan-binding lectin (MBL), the first component of the lectin pathway of complement activation. Furthermore, loss of the terminal glucose units on the derivatized lysine residues in mouse embryonic fibroblasts lacking the LH3 protein leads to defective disulphide bonding and oligomerization of rat MBL-A, with a decrease in the proportion of the larger functional MBL oligomers. The oligomerization could be completely restored with the full length LH3 or the amino-terminal fragment of LH3 that possesses the glycosyltransferase activities. Our results confirm that LH3 is the only enzyme capable of glucosylating the galactosylhydroxylysine residues in proteins with a collagenous domain. In mice lacking the lysyl hydroxylase activity of LH3, but with untouched galactosyltransferase and glucosyltransferase activities, reduced circulating MBL-A levels were observed. Oligomerization was normal, however and residual lysyl hydroxylation was compensated in part by other lysyl hydroxylase isoenzymes. Our data suggest that LH3 is commonly involved in biosynthesis of collagenous proteins and the glucosylation of galactosylhydroxylysines residues by LH3 is crucial for the formation of the functional high-molecular weight MBL oligomers.
Lysyl hydroxylase 3 (LH3) has lysyl hydroxylase, galactosyltransferase, and glucosyltransferase a... more Lysyl hydroxylase 3 (LH3) has lysyl hydroxylase, galactosyltransferase, and glucosyltransferase activities, which are sequentially required for the formation of glucosylgalactosyl hydroxylysines in collagens. Here we demonstrate for the first time that LH3 also modifies the lysine residues in the collagenous domain of adiponectin, which has important roles in glucose and lipid metabolism and inflammation. Hydroxylation and, especially, glycosylation of the lysine residues of adiponectin have been shown to be essential for the formation of the more active high molecular weight adiponectin oligomers and thus for its function. In cells that totally lack LH3 enzyme, the galactosylhydroxylysine residues of adiponectin were not glucosylated to glucosylgalactosylhydroxylysine residues and the formation of high and middle molecular weight adiponectin oligomers was impaired. Circulating adiponectin levels in mutant mice lacking the lysyl hydroxylase activity of LH3 were significantly reduced, which indicates that LH3 is required for complete modification of lysine residues in adiponectin and the loss of some of the glycosylated hydroxylysine residues severely affects the secretion of adiponectin. LH mutant mice with reduced adiponectin level showed a high fat diet-induced increase in glucose, triglyceride, and LDLcholesterol levels, hallmarks of the metabolic syndrome in humans. Our results reveal the first indication that LH3 is an important regulator of adiponectin biosynthesis, secretion and activity and thus might be a potential candidate for therapeutic applications in diseases associated with obesity and insulin resistance.
Lysyl hydroxylases (LH), which catalyze the post-translational modifications of lysines in collag... more Lysyl hydroxylases (LH), which catalyze the post-translational modifications of lysines in collagen and collagen-like proteins, function as dimers. However, the amino acids responsible for dimerization and the role of dimer formation in the enzymatic activities of LH have not yet been identified. We have localized the region responsible for the dimerization of lysyl hydroxylase 3 (LH3), a multifunctional enzyme of collagen biosynthesis, to a sequence of amino acids between the glycosyltransferase activity and the lysyl hydroxylase activity domains. This area is covered by amino acids 541-547 in human LH3, but contains no cysteine residues. The region is highly conserved among LH isoforms, and is also involved in the dimerization of LH1 subunits. Dimerization is required for the LH activity of LH3, whereas it is not obligatory for the glycosyltransferase activities. In order to determine whether complex formation can occur between LH molecules originating from different species, and between different LH isoforms, double expressions were generated in a baculovirus system. Heterocomplex formation between mouse and human LH3, between human LH1 and LH3 and between human LH2 and LH3 was detected by western blot analyses. However, due to the low amount of complexes formed, the in vivo function of heterocomplexes remains unclear.
Introduction: Oral tongue squamous cell carcinoma (OTSCC) is the most common cancer of the oral c... more Introduction: Oral tongue squamous cell carcinoma (OTSCC) is the most common cancer of the oral cavity. Contradictory results have been observed on the involvement of herpes simplex virus 1 (HSV-1) in oral squamous cell carcinomas. Here, we aimed to study the predominance of HSV-1 or HSV-2 in oral HSV infections and to investigate the presence of HSV-1 in OTSCC and its effect on carcinoma cell viability and invasion.Methods: The distribution of HSV types one and two in diagnostic samples taken from suspected oral HSV infections was determined from the Helsinki University Hospital Laboratory database. We then analysed 67 OTSCC samples for HSV-1 infection using immunohistochemical staining. We further tested the effects of HSV-1 using six concentrations (0.00001–1.0 multiplicity of infection [MOI]) on viability and two concentrations (0.001 and 0.1 MOI) on invasion of highly invasive metastatic HSC-3 and less invasive primary SCC-25 OTSCC cell lines using MTT and Myogel-coated Transwe...
ObjectiveAlthough there have been remarkable achievements in the molecular landscape of oral squa... more ObjectiveAlthough there have been remarkable achievements in the molecular landscape of oral squamous cell carcinoma (OSCC) in recent years, bringing advances in the understanding of its pathogenesis, development and progression, little has been applied in the prognosis and choosing the optimal treatment. In this study, we explored the influence of the stress induced phosphoprotein 1 (STIP1), which is frequently reported to be highly expressed in many cancers, in OSCCs.MethodsSTIP1 expression was assessed in the TCGA database and in two independent cohorts by immunohistochemistry. Knockdown strategy was applied in OSCC cell lines to determine the impact of STIP1 on viability, proliferation, migration and invasion. The zebrafish model was applied for studying tumor formation and metastasis in vivo. The association of STIP1 and miR-218-5p was explored by bioinformatics and mimics transfection.ResultsSTIP1 was highly expressed in OSCCs and significantly associated with shortened surviv...
Proper functioning of each secretory and endocytic compartment relies on its unique pH micro-envi... more Proper functioning of each secretory and endocytic compartment relies on its unique pH micro-environment that is known to be dictated by the rates of V-ATPase-mediated H+ pumping and its leakage back to the cytoplasm via an elusive “H+ leak” pathway. Here, we show that this proton leak across Golgi membranes involves AE2a (SLC4A2a)-mediated bicarbonate-chloride exchange, as it is strictly dependent on both bicarbonate import (in exchange of chloride export) and the AE2a expression level in the cells. Imported bicarbonate anions and luminal protons then facilitate a common buffering reaction that yields carbon dioxide and water before their egress back to the cytoplasm via diffusion or water channels. The high surface-volume ratio of flattened Golgi cisternae helps this process, as their shape is optimal for water and gas exchange. Interestingly, this pathway is often upregulated in cancers and established cancer cell lines, and responsible for their markedly elevated Golgi resting p...
Conventional chemotherapeutic agents are nonselective, often resulting in severe side effects and... more Conventional chemotherapeutic agents are nonselective, often resulting in severe side effects and the development of resistance. Therefore, new molecular-targeted therapies are urgently needed to be integrated into existing treatment regimens. Here, we performed a high-throughput compound screen to identify a synergistic interaction between ionizing radiation and 396 anticancer compounds. The assay was run using five human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) cell lines cultured on the human tumor-derived matrix Myogel. Our screen identified several compounds with strong synergistic and antagonistic effects, which we further investigated using multiple irradiation doses. Navitoclax, which emerged as the most promising radiosensitizer, exhibited synergy with irradiation regardless of the p53 mutation status in all 13 HNSCC cell lines. We performed a live cell apoptosis assay for two representative HNSCC cell lines to examine the effects of navit...
Cancer invasiveness and metastatic spread are the main causes of cancer-associated deaths, yet su... more Cancer invasiveness and metastatic spread are the main causes of cancer-associated deaths, yet successful therapies for their prevention are currently non-existing. Here we exploit cancer cell reprogramming, a strategy developed earlier to prevent tumour growth, to show that cancer cell invasiveness can be reversed via restoring their Golgi pH homeostasis and normal glycosylation status. Specifically, we demonstrate that AE2a (SLC4A2a)-mediated bicarbonate-chloride exchange controls proton leakage across Golgi membranes via a well-known buffering reaction that produces water and carbon dioxide from luminal protons and bicarbonate anions. Its forced overexpression in non-malignant cells, or upregulation in certain cancer cells in vitro and in vivo, also coincides with elevated Golgi resting pH, the synthesis of invasion and metastasis-promoting glycans and a shorter life span of cancer patients. Finally, AE2 knockdown in cancer cells blocked their invasiveness via restoring their Gol...
Abstract Lysyl hydroxylase (LH) catalyzes the post-translational formation of hydroxylysines in c... more Abstract Lysyl hydroxylase (LH) catalyzes the post-translational formation of hydroxylysines in collagens and collagenous proteins. Three lysyl hydroxylase isoforms, LH1, LH2 and LH3, have been identified from different species. In addition, LH2 has two alternatively spliced forms, LH2a and LH2b. The hydroxylysines have an important role in the formation of the intermolecular collagen crosslinks that stabilize the collagen fibrils. Some of the hydroxylysine residues are further glycosylated. In this thesis the substrate amino acid sequence specificities of the LH isoforms were analyzed using synthetic peptide substrates. The data did not indicate strict amino acid sequence specificity for the LH isoforms. However, there seemed to be a preference for some sequences to be bound and hydroxylated by a certain isoform. Galactosylhydroxylysyl glucosyltransferase (GGT) catalyzes the formation of glucosylgalactosylhydroxylysine. In this study, LH3 was shown to be a multifunctional enzyme, possessing LH and GGT activities. The DXD-like motif, characteristic of many glycosyltransferase families, and the conserved cysteine and leucine residues in the N-terminal part of the LH3 molecule were critical for the GGT activity, but not for the LH activity of the molecule. The GGT/LH3 protein level was found to be decreased in skin fibroblasts and in the culture media of cells collected from members of a Finnish epidermolysis bullosa simplex (EBS) family, which was earlier reported to have a deficiency of GGT activity. In this study, we showed that the reduction of enzyme activity is not due to a mutation or lower expression of the LH3 gene. Our data indicate that the decreased GGT/LH3 activity in cells has an effect on the deposition and organization of the key extracellular matrix components, collagen types VI and I and fibronectin, and these changes are transmitted to the cytoskeletal network. These findings underline LH3 as an important extracellular regulator
Oral carcinoma is one of the most aggressive cancers, and despite the advances in the therapy, it... more Oral carcinoma is one of the most aggressive cancers, and despite the advances in the therapy, its mortality is still high. An attention in cancer treatment has focused on natural compounds due to their potential beneficial effects on human health. In this study, the effects of dietary supplement Fermented Wheat Germ Extract (FWGE) on oral tongue squamous cell carcinoma (OTSCC) cells were investigated in vitro using three cell lines (HSC-3, SAS, SCC-25) with variable aggressiveness. The cell viability was significantly decreased by the treatment with high concentration of FWGE in every cell line. Regarding migration and invasion, HSC-3 and SCC-25 cells were most sensitive to FWGE since their movement was significantly reduced with 5 and 10 mg/ml FWGE, while SAS was inhibited only with 10 mg/ml FWGE. Chemotherapeutic compounds (cisplatin and 5-fluorouracil) significantly reduced all OTSCC cells viability. Importantly, combination of these drugs with 10 mg/ml FWGE significantly decreased the cell viability compared to the treatment with the chemotherapeutics or FWGE alone. Based on these in vitro experiments, the use of FWGE seems to improve the anticancer effects on OTSCC cells. Further in vivo and clinical studies should be conducted to verify the positive effects of FWGE for OTSCC patients.
Proper functioning of each secretory and endocytic compartment relies on its unique pH micro-envi... more Proper functioning of each secretory and endocytic compartment relies on its unique pH micro-environment that is known to be dictated by the rates of V-ATPase-mediated H+ pumping and its leakage back to the cytoplasm via an elusive “H+ leak” pathway. Here, we show that this proton leak across Golgi membranes is mediated by the AE2a (SLC4A2a)-mediated bicarbonate-chloride exchange, as it is strictly dependent on bicarbonate import (in exchange for chloride export) and the expression level of the Golgi-localized AE2a anion exchanger. In the acidic Golgi lumen, imported bicarbonate anions and protons then facilitate a common buffering reaction that yields carbon dioxide and water before their egress back to the cytoplasm via diffusion or water channels. The flattened morphology of the Golgi cisternae helps this process, as their high surface-volume ratio is optimal for water and gas exchange. Interestingly, this net acid efflux pathway is often upregulated in cancers and established ca...
Oral tongue squamous cell carcinoma (OTSCC), the most common cancer in the oral cavity, is aggres... more Oral tongue squamous cell carcinoma (OTSCC), the most common cancer in the oral cavity, is aggressive and its incidence is increasing globally. Human host defense cationic antimicrobial peptide-18/antimicrobial peptide leucine-leucine-37 (hCAP18/LL-37) plays a complex role in various types of cancers. In the present study, we characterized the effects of exogenous LL-37 on three OTSCC cell lines and determined the expression of hCAP18/LL-37 in oral dysplastic and OTSCC patient samples. Our data revealed that LL-37, especially in high doses, mostly reduced the proliferation of OTSCC cells, but the effect was fluctuating. However, LL-37 stimulated the migration and invasion of OTSCC cells. The high dose of LL-37 also increased the amount of total epidermal growth factor receptor (EGFR) probably due to stabilization of the receptor to the plasma membrane. However, activation of EGFR downstream pathways was mostly decreased. Our immunohistochemical analysis showed that the hCAP18/LL-37 expression was higher in normal/mild dysplasia than in moderate/severe dysplasia and OTSCC. The hCAP18/LL-37 expression did not correlate with clinicopathological features or outcome of OTSCC patients. Our data suggest that LL-37 has a fluctuating effect on proliferation, migration and invasion of OTSCC cells, but it does not seem to play a role in the progression of OTSCC.
In vitro cancer drug testing carries a low predictive value. We developed the human leiomyoma–der... more In vitro cancer drug testing carries a low predictive value. We developed the human leiomyoma–derived matrix “Myogel” to better mimic the human tumor microenvironment (TME). We hypothesized that Myogel could provide an appropriate microenvironment for cancer cells, thereby allowing more in vivo–relevant drug testing. We screened 19 anticancer compounds, targeting the epidermal growth factor receptor (EGFR), MEK, and PI3K/mTOR on 12 head and neck squamous cell carcinoma (HNSCC) cell lines cultured on plastic, mouse sarcoma–derived Matrigel (MSDM), and Myogel. We applied a high-throughput drug screening assay under five different culturing conditions: cells in two-dimensional (2D) plastic wells and on top or embedded in Matrigel or Myogel. We then compared the efficacy of the anticancer compounds to the response rates of 19 HNSCC monotherapy clinical trials. Cancer cells on top of Myogel responded less to EGFR and MEK inhibitors compared to cells cultured on plastic or Matrigel. Howev...
Pro-tumorigenic activities of matrix metalloproteinase (MMP) 9 have been linked to many cancers, ... more Pro-tumorigenic activities of matrix metalloproteinase (MMP) 9 have been linked to many cancers, but recently the tumour-suppressing role of MMP9 has also been elucidated. The multifaceted evidence on this subject prompted us to examine the role of MMP9 in the behaviour of oral tongue squamous cell carcinoma (OTSCC) cells. We used gelatinase-specific inhibitor, CTT2, and short hairpin (sh) RNA gene silencing to study the effects of MMP9 on proliferation, motility and invasion of an aggressive OTSCC cell line, HSC-3. We found that the migration and invasion of HSC-3 cells were increased by CTT2 and shRNA silencing of MMP9. Proliferation, in turn, was decreased by MMP9 inhibition. Furthermore, arresten-overexpressing HSC-3 cells expressed increased levels of MMP9, but exhibited decreased motility compared with controls. Interestingly, these cells restored their migratory capabilities by CTT2 inhibition of MMP9. Hence, although higher MMP9 expression could give rise to an increased tumour growth in vivo due to increased proliferation, in some circumstances, it may participate in yet unidentified molecular mechanisms that reduce the cell movement in OTSCC. However, MMPs have far more complex roles in cancer, and some MMPs are now also shown to suppress some aspects of cancer progression (3). MMP9 has conventionally been considered as a pro-tumorigenic enzyme in oral squamous cell carcinoma (OSCC). A recent meta-analysis study showed that MMP9 overexpression is a predictor of poor prognosis in OTSCC patients (4). However, whether MMP9 expression in the most aggressive tumours enhances tumour progression or is more a consequence of tumour aggressiveness is unknown. Previous studies have revealed both pro-and anti-tumorigenic roles of MMP9 (reviewed in detail by Vilen et al. 2013), and its role in oral cancer is far from clear (5). Jordan et al. (2004) found that the mRNA levels of MMP9 were significantly higher in oral dysplasias that progressed to oral cancer than in those that did not (6). Pro-and active forms, total activities and the activation ratio of MMP9 were also significantly elevated in OSCC samples compared with their adjacent areas histologically rated as normal tissues (7). In contrast, Stokes et al. (2010) showed that MMP9 mRNA in primary tumours compared with adjacent peritumoral tissues was significantly decreased in head and neck squamous cell carcinomas with lymph node metastasis compared with non-metastatic tumours (8). Moreover, Lin et al. (2012) demonstrated that although patients with OSCC exhibit significantly higher levels of MMP9 than healthy controls, and even though MMP9 plasma levels are associated with more advanced clinical stages, MMP9 level was not associated with positive lymph node or distal metastasis (9). Evidence for the oncosuppressive role of MMP9 has also emerged for other cancers such as colitis-associated cancers (10) and breast cancer when MMP9 is produced by cancer cells (11). Recently, we have reported increased MMP9 expression in HSC-3 clone overexpressing MMP8 with decreased migration and invasion (12). Additionally, although anti-MMP2 and-9 peptides inhibit tongue carcinoma growth (13,14) and angiogenesis (13), they do not prevent the spread of carcinoma 4 cells in nude mice (14). Instead, when anti-MMP2 and-9 peptides were used in combination with proMMP9 targeting therapy, increased HSC-3 tumour growth was observed in mice (14). This study aims to elucidate the role of MMP9 in the behaviour of aggressive oral tongue carcinoma cells in vitro by using various genetically modified cell models with altered MMP9 expression as well as inhibition of MMP9 by gelatinase (MMP2 and-9) inhibitor peptide CTT2 (15). Materials and methods Cell Culture Native cell lines: Human tongue squamous cell carcinoma cell lines HSC-3 (Japanese Collection of Research Bioresources (JCRB) Cell Bank, JCRB0623), SAS (JCRB Cell Bank, JCRB0260), SCC-25 (American Type Culture Collection (ATCC), CRL 1628), human malignant melanoma G361 (ATCC, CRL-1424) and human Caucasian breast adenocarcinoma cell line MDA-MB-231 (ATCC, HTB-26) were cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12, 1:1 (Gibco) supplemented with 10% heat-inactivated foetal bovine serum (FBS, Gibco), 50 μg/ml ascorbic acid, 100 U/ml penicillin, 100 μg/ml streptomycin, 250 ng/ml amphotericin B and 0.4 µg/ml hydrocortisone (all from Sigma-Aldrich). Human embryonic kidney cell HEK-293 (ATCC, CRL-1573), human gingival fibroblasts (GFs) and human spontaneously immortalized keratinocytes (HaCaT) (16) were cultured in DMEM (Sigma-Aldrich) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml ascorbic acid, 250 ng/ml amphotericin B and 1 mM sodium pyruvate (all from Sigma-Aldrich). GFs used in this study were obtained from biopsies of healthy gingiva as described earlier (17). The human papillomavirus HPV16 immortalized human oral epithelial cells (IHGK) (18) were cultured in Keratinocyte-SFM (Gibco) supplemented with 5 ng/ml human recombinant epidermal growth factor, 50 µg/ml bovine pituitary extract (both from Gibco), 100 U/ml
Chemoradiation is an established approach in the treatment of advanced oral tongue squamous cell ... more Chemoradiation is an established approach in the treatment of advanced oral tongue squamous cell carcinoma (OTSCC), but therapy may cause severe side-effects due to signal interchanges between carcinoma and the tumour microenvironment (TME). In this study, we examined the potential use of our human 3D myoma disc and Myogel models in in vitro chemoradiation studies by analysing the effects of ionizing radiation (IR) and the combined effect of heparanase I (HPSE1) inhibitors and IR on OTSCC cell proliferation, invasion and MMP-2 and - 9 production. Finally, we analysed the long-term effects of IR by studying clones of previously irradiated and invaded HSC-3 cells. We found that in both human uterine leiomyoma-based extracellular matrix models IR inhibited the invasion of HSC-3 cells, but blocking HPSE1 activity combined with IR induced their invasion. Low doses of IR increased MMP expression and initiated epithelial-mesenchymal transition in cells cultured on myoma discs. We conclude ...
Sepsis delays wound re-epithelialization. In this study we explored the effect of human sepsis se... more Sepsis delays wound re-epithelialization. In this study we explored the effect of human sepsis sera as well as the effects of cytokines, growth factors and exosomes of sepsis sera treated normal fibroblasts (NF) on keratinocyte migration and proliferation in vitro. Serum samples were taken on days 1, 4, and 9 from 44 patients diagnosed with severe sepsis, and from 14 matching healthy controls. We evaluated the effects of sepsis serum with or without TNF-α, EGF, EGF receptor inhibitor or exosomes of sepsis sera treated NF on human keratinocyte (HaCaT) proliferation (BrdU assay), viability (MTT assay), and migration (horizontal wound healing model). Cytokine levels of sepsis and healthy sera were measured by multiplex assay. Comparisons between groups were carried out using SPSS statistics and P < 0.05 was considered significant. Severe-sepsis sera collected on days 1, 4, and 9 reduced keratinocyte proliferation by 6% (P = 0.005), 20% (P = 0.001), and 18% (P = 0.002), respectively,...
Post-translational modifications are necessary for collagen precursor molecules (procollagens) to... more Post-translational modifications are necessary for collagen precursor molecules (procollagens) to acquire final shape and function. However, the mechanism and contribution of collagen modifications that occur outside the endoplasmic reticulum and Golgi are not understood. We discovered that VIPAR, with its partner proteins, regulate sorting of lysyl hydroxylase 3 (LH3, also known as PLOD3) into newly identified post-Golgi collagen IV carriers and that VIPAR-dependent sorting is essential for modification of lysines in multiple collagen types. Identification of structural and functional collagen abnormalities in cells and tissues from patients and murine models of the autosomal recessive multisystem disorder Arthrogryposis, Renal dysfunction and Cholestasis syndrome caused by VIPAR and VPS33B deficiencies confirmed our findings. Thus, regulation of post-Golgi LH3 trafficking is essential for collagen homeostasis and for the development and function of multiple organs and tissues.
<p>(A) Amount of MBL-A was reduced in the serum of LH mutant mice (MUT) compared with the c... more <p>(A) Amount of MBL-A was reduced in the serum of LH mutant mice (MUT) compared with the corresponding wild type mice (WT). Amount of MBL-A was analyzed from the serum of the 2 months old male (n = 4 WT, 4 MUT) and female (n = 4 WT, 4 MUT) and 1 year old male (n = 4 WT, 3 MUT) and female (n = 5 WT, 6 MUT) by immunoblots. The values represent the average ± SD of the samples. MBL-A monomers in the serum of 2 months old female (B) and 1 year old male (C) wild type and LH mutant mice did not show significant differences in electrophoretic mobility. (D) The immunoblot analysis of liver homogenate (40 µg of soluble protein) of wild type and LH mutant male mice did not show differences in mobility of MBL-A monomers. (E) The amount of MBL-A in liver of LH mutant mice is higher than in wild type. Amount of MBL-A in liver was analyzed with immunoblots and α-tubulin was used as control to normalize the quantities of protein. In immunoblots B, C and D two representative wild type and LH samples are shown. MBL-A in LH mutant mice does not show doublet band, differing from the results of rat MBL-A overexpressed in cell culture system (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113498#pone-0113498-g001" target="_blank">Fig. 1A</a>). The values in figure A and E represent the average ± SD of the samples. P values were calculated using unpaired homoscedastic student t-test with two-tailed distribution. * p<0.05.</p
Lysyl hydroxylase 3 (LH3) is a multifunctional protein with lysyl hydroxylase, galactosyltransfer... more Lysyl hydroxylase 3 (LH3) is a multifunctional protein with lysyl hydroxylase, galactosyltransferase and glucosyltransferase activities. The LH3 has been shown to modify the lysine residues both in collagens and also in some collagenous proteins. In this study we show for the first time that LH3 is essential for catalyzing formation of the glucosylgalactosylhydroxylysines of mannan-binding lectin (MBL), the first component of the lectin pathway of complement activation. Furthermore, loss of the terminal glucose units on the derivatized lysine residues in mouse embryonic fibroblasts lacking the LH3 protein leads to defective disulphide bonding and oligomerization of rat MBL-A, with a decrease in the proportion of the larger functional MBL oligomers. The oligomerization could be completely restored with the full length LH3 or the amino-terminal fragment of LH3 that possesses the glycosyltransferase activities. Our results confirm that LH3 is the only enzyme capable of glucosylating the galactosylhydroxylysine residues in proteins with a collagenous domain. In mice lacking the lysyl hydroxylase activity of LH3, but with untouched galactosyltransferase and glucosyltransferase activities, reduced circulating MBL-A levels were observed. Oligomerization was normal, however and residual lysyl hydroxylation was compensated in part by other lysyl hydroxylase isoenzymes. Our data suggest that LH3 is commonly involved in biosynthesis of collagenous proteins and the glucosylation of galactosylhydroxylysines residues by LH3 is crucial for the formation of the functional high-molecular weight MBL oligomers.
Lysyl hydroxylase 3 (LH3) has lysyl hydroxylase, galactosyltransferase, and glucosyltransferase a... more Lysyl hydroxylase 3 (LH3) has lysyl hydroxylase, galactosyltransferase, and glucosyltransferase activities, which are sequentially required for the formation of glucosylgalactosyl hydroxylysines in collagens. Here we demonstrate for the first time that LH3 also modifies the lysine residues in the collagenous domain of adiponectin, which has important roles in glucose and lipid metabolism and inflammation. Hydroxylation and, especially, glycosylation of the lysine residues of adiponectin have been shown to be essential for the formation of the more active high molecular weight adiponectin oligomers and thus for its function. In cells that totally lack LH3 enzyme, the galactosylhydroxylysine residues of adiponectin were not glucosylated to glucosylgalactosylhydroxylysine residues and the formation of high and middle molecular weight adiponectin oligomers was impaired. Circulating adiponectin levels in mutant mice lacking the lysyl hydroxylase activity of LH3 were significantly reduced, which indicates that LH3 is required for complete modification of lysine residues in adiponectin and the loss of some of the glycosylated hydroxylysine residues severely affects the secretion of adiponectin. LH mutant mice with reduced adiponectin level showed a high fat diet-induced increase in glucose, triglyceride, and LDLcholesterol levels, hallmarks of the metabolic syndrome in humans. Our results reveal the first indication that LH3 is an important regulator of adiponectin biosynthesis, secretion and activity and thus might be a potential candidate for therapeutic applications in diseases associated with obesity and insulin resistance.
Lysyl hydroxylases (LH), which catalyze the post-translational modifications of lysines in collag... more Lysyl hydroxylases (LH), which catalyze the post-translational modifications of lysines in collagen and collagen-like proteins, function as dimers. However, the amino acids responsible for dimerization and the role of dimer formation in the enzymatic activities of LH have not yet been identified. We have localized the region responsible for the dimerization of lysyl hydroxylase 3 (LH3), a multifunctional enzyme of collagen biosynthesis, to a sequence of amino acids between the glycosyltransferase activity and the lysyl hydroxylase activity domains. This area is covered by amino acids 541-547 in human LH3, but contains no cysteine residues. The region is highly conserved among LH isoforms, and is also involved in the dimerization of LH1 subunits. Dimerization is required for the LH activity of LH3, whereas it is not obligatory for the glycosyltransferase activities. In order to determine whether complex formation can occur between LH molecules originating from different species, and between different LH isoforms, double expressions were generated in a baculovirus system. Heterocomplex formation between mouse and human LH3, between human LH1 and LH3 and between human LH2 and LH3 was detected by western blot analyses. However, due to the low amount of complexes formed, the in vivo function of heterocomplexes remains unclear.
Uploads
Papers by Maija Risteli