Expression of class III β-tubulin in normal and neoplastic human tissues

 The class III β-tubulin isotype is widely used as a neuronal marker in normal and neoplastic tissues. This isotype was, however, also immunodetected in certain tumours of non-neuronal origin such as squamous cell carcinoma. Using a newly described monoclonal antibody we compared the distribution of class III β-tubulin in normal and neoplastic tissues. The TU-20 mouse monoclonal antibody was prepared against a conserved synthetic peptide from the C-terminus of the human class III β-tubulin isotype, and its specificity was confirmed by immunoblotting, by competitive enzyme-linked immunosorbent assay and by immunofluorescence microscopy on cultured cells. In different cell lines of various origins the antibody reacted only with neuroblastoma Neuro-2a cells and with embryonal carcinoma P19 cells stimulated to neuronal differentiation by retinoic acid. Immunohistochemistry on formaldehyde-fixed paraffin-embedded normal human tissues revealed the presence of the class III β-tubulin isotype in cell bodies and processes of neuronal cells in the peripheral and central nervous systems. In other tissues, this β-tubulin isotype was not immunodetected. Class III β-tubulin was found in all cases of ganglioneuroblastoma, ganglioneuroma, medulloblastoma, neuroblastoma, sympathoblastoma and in one case of teratoma. In contrast, no reactivity was detected in tumours of non-neuronal origin, including 32 cases of squamous cell carcinoma. The results indicate a specific TU-20 epitope expression exclusively in neuronal tissues. The antibody could thus be a useful tool for the probing of class III β-tubulin functions in neurons as well as for immunohistochemical characterisation of tumours of neuronal origin. Accepted: 29 July 1997     

Class III β-tubulin,a novel biomarker in the human melanocyte lineage

It is generally thought that class III β-tubulin expression is limited to cells of the neural lineage and is therefore often used to identify neurons amongst other cell types, both in vivo and in vitro. Melanocytes are derived from the neural crest and share both morphological features and functional characteristics with peripheral neurons. Here, we show that these similarities extend to class III β-tubulin (TUBB3) expression, and that human melanocytes express this protein both in vivo and in vitro. In addition, we studied the expression of class III β-tubulin in two murine melanogenic cell lines and show that expression of this protein starts as melanoblasts mature into melanocytes. Melanin bleaching experiments revealed close proximity between melanin and TUBB3 proteins. In vitro stimulation of primary human melanocytes by α-MSH indicated separate regulatory mechanisms for melanogenesis and to TUBB3 expression. Together, these observations imply that human melanocytes express TUBB3 and that this protein should be recognized as a wider marker for multiple neural crest-derived cells.     

Effect of melatonin on β-tubulin and MAP2 expression in NIE-115 cells

Physiological concentrations of the pineal hormone melatonin induce an increase of microtubules in neuroblastoma NIE-115 cells. This effect is due to an increase in the polymerization state of tubulin. Concomitantly, higher levels of soluble β-tubulin are present in the treated cells. Unexpectedly, no significant changes in the levels of β-tubulin or its mRNA occur in the presence of melatonin reflecting perhaps a strict control of its steady state in a physiological context. In contrast, higher amounts of microtubule-associated-protein 2 are found when the cells are exposed to melatonin. These findings support the idea that tubulin polymerization process is one of the targets of melatonin action. Furthermore, our results might explain the increase in the length and number of neurites present in these cells when they are treated with this hormone.     

Localization ofα 2-macroglobulin in human primary sarcomas and synthesis in established cell linesin human primary sarcomas and synthesis in established cell lines

Summary The presence ofα ₂-macroglobulin was detected with the avidin-biotin technique in more than 20-yr-old paraffin blocks from human sarcomas.α ₂M was found mainly in the cytoplasm of the tumor cells, and almost all tumor cells were positive. This serum glycoprotein, which is a major plasma proteinase inhibitor with a wide specificity, was also shown to be synthesized and secreted by all three cell lines derived from primary sarcomas but was not detected in cultures of the autologous skin fibroblasts. For the detection ofα ₂M in situ and in vitro an antiserum to tumor-associatedα ₂-macroglobulin was used. Our work was supported by grant no. 55-B86-21XB, from the Swedish Cancer Society.     

Role of the mitochondria in immune-mediated apoptotic death of the human pancreatic β cell line βLox5

Mitochondria are indispensable in the life and death of many types of eukaryotic cells. In pancreatic beta cells, mitochondria play an essential role in the secretion of insulin, a hormone that regulates blood glucose levels. Unregulated blood glucose is a hallmark symptom of diabetes. The onset of Type 1 diabetes is preceded by autoimmune-mediated destruction of beta cells. However, the exact role of mitochondria has not been assessed in beta cell death. In this study, we examine the role of mitochondria in both Fas- and proinflammatory cytokine-mediated destruction of the human beta cell line, βLox5. IFNγ primed βLox5 cells for apoptosis by elevating cell surface Fas. Consequently, βLox5 cells were killed by caspase-dependent apoptosis by agonistic activation of Fas, but only after priming with IFNγ. This beta cell line undergoes both apoptotic and necrotic cell death after incubation with the combination of the proinflammatory cytokines IFNγ and TNFα. Additionally, both caspase-dependent and -independent mechanisms that require proper mitochondrial function are involved. Mitochondrial contributions to βLox5 cell death were analyzed using mitochondrial DNA (mtDNA) depleted βLox5 cells, or βLox5 ρ(0) cells. βLox5 ρ(0) cells are not sensitive to IFNγ and TNFα killing, indicating a direct role for the mitochondria in cytokine-induced cell death of the parental cell line. However, βLox5 ρ(0) cells are susceptible to Fas killing, implicating caspase-dependent extrinsic apoptotic death is the mechanism by which these human beta cells die after Fas ligation. These data support the hypothesis that immune mediators kill βLox5 cells by both mitochondrial-dependent intrinsic and caspase-dependent extrinsic pathways.     

Isothiocyanate–drug interactions in the human adenocarcinoma cell line Caco-2

Loss or mutation of the PTEN (phosphatase and tensin homologue deleted on chromosome 10) gene is associated with resistance to epidermal growth factor receptor (EGFR) inhibitors. However, the mechanism underlying remains elusive. In this study, we aimed to explore whether sensitivity to the EGFR tyrosine kinase inhibitor (TKI) is affected by PTEN status in endometrial cancer cells. PTEN siRNA and the PTEN gene were transfected into HEC-1A and Ishikawa endometrial cancer cells using lentiviral vectors. Cells were treated under various concentrations of RG14620 and rapamycin, which are EGFR and mammalian target of rapamycin (mTOR) inhibitors, respectively. The IC(50) of RG16420 was determined by using the MTT method. Cell apoptosis and the cell cycle were studied, and activation of EGFR, AKT, and p70S6 were detected by Western blot analysis. Loss of PTEN promoted cell proliferation and led to significant increases in the levels of EGFR, phospho-EGFR, AKT, phospho-AKT, and phospho-mTOR proteins. Ishikawa and HEC-1A(PTENkd) cells that displayed loss and inactivation of PTEN function were resistant to RG14620. HEC-1A and Ishikawa(PTEN) cells with intact PTEN were sensitive to RG14620. The combination of two inhibitors was more effective than both monotherapies, particularly in carcinoma cells with PTEN dysfunction. Decreased phospho-EGFR protein expression was observed in all cell lines that were sensitive to RG14620. Decreased phospho-AKT and phospho-p70S6 protein expression was observed in PTEN-intact cells that were sensitive to RG14620. PTEN loss results in resistance to EGFR TKI, which was reversed by PTEN reintroduction or mTOR inhibitor treatment. The combined treatment of EGFR TKI and the mTOR inhibitor provided a synergistic effect by promoting cell death in PTEN-deficient and PTEN-intact endometrial cancer cells, particularly in PTEN-deficient carcinoma cells with up-regulated EGFR activation.     

Frequent loss of heterozygosity in the β2-microglobulin region of chromosome 15 in primary human tumors

Downregulation or total loss of HLA class I expression on tumor cells is known as a mechanism of cancer immune escape. Alterations of the HLA phenotype are frequently due to mutations affecting genes encoding the HLA class I heavy chains located on chromosome 6p21 or the β2-microglobulin (β2m) gene encoding the light chain of the HLA complex located on chromosome 15q21. Frequently irreversible total loss of HLA class I molecules is due to the coincidence of two molecular events, the mutation of one β2m gene and the loss of the second copy. The latter is detectable as loss of heterozygosity (LOH) of microsatellite markers in the β2m region on chromosome 15q21 (LOH-15q21). Thus, LOH-15q21 might be an important event in the processes of HLA class I downregulation and total loss. Here we studied the frequency of LOH-15q21 in tumor tissues of different entities. By determining the status of heterozygosity of two microsatellite markers we detected LOH-15q21 in 44% of bladder carcinomas (n = 69), in 35% of colon carcinomas (n = 95), in 16% of melanomas (n = 70) but only in 7% of renal cancers (n = 45). Moreover, we observed a frequent coincidence of LOH-15q21 and LOH-6p21 in colorectal carcinoma, bladder carcinoma and melanoma, but not for renal carcinoma. We believe that the high incidence of LOH-15q21 in some malignancies and especially the coincidence of LOH-15q21 and LOH-6p21 might have a strong impact on tumor immunogenicity and on the efficiency of cancer immunotherapy.     

Class III β-tubulin and the cytoskeletal gateway for drug resistance in ovarian cancer

The Class III β-tubulin isotype (βIII-tubulin) is a predictive biomarker in ovarian cancer and other solid tumor malignancies. We discovered that βIII-tubulin function is linked to two GTPases: guanylate-binding protein 1 (GBP1), which activates its function, and GNAI1, which inhibits it. This finding was demonstrated in a panel of ovarian cancer cells resistant to several chemotherapeutic agents. Using a protein microarray, we identified PIM1 as the downstream partner of GBP1, recruited into the cytoskeleton under hypoxic conditions. The clinical value of these observations was tested by performing an archive study of 98 ovarian cancer patients, which demonstrated that the βIII-tubulin -/PIM1- cohort responded to treatment, exhibiting long overall survival (OS), while βIII-tubulin +/PIM+ patients experienced poor outcomes and OS times similar to patients receiving palliation alone. βIII-tubulin expression is commonly believed responsible for paclitaxel resistance due to its enhancement of the dynamic instability of microtubules, which counteracts the activity of taxanes. In contrast, our research reveals that βIII-tubulin behaves as a gateway for prosurvival signals, such as PIM1, to move into the cytoskeleton. When cells are exposed to microenvironmental stressors, they activate this pathway by telling the cytoskeleton to incorporate PIM1 through GBP1 and βIII-tubulin, which ultimately leads to drug resistance. This discovery reveals that βIII-tubulin does not act alone but requires partners to play its role. The discovery of such protein:protein interactions underlying this prosurvival cascade makes feasible the development of therapeutic approaches using novel compounds that are capable of inhibiting the transmission of prosurvival signals into the cytoskeleton.     

The effects of estrogen receptors α- and β-specific agonists and antagonists on cell proliferation and energy metabolism in human bone cell line

In cultured human osteoblasts estradiol-17β (E2) modulated DNA synthesis, the specific activity of creatine kinase BB (CK), 12 and 15 lipoxygenase (LO) mRNA expression and formation of 12- and 15-hydroxyeicosatetraenoic acid (HETE). We now investigate the response of human bone cell line (SaOS2) to phytoestrogens and estrogen receptors (ER)-specific agonists and antagonists. Treatment of SaSO2 with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ-specific agonist), 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα-specific agonist), biochainin A (BA), daidzein (D), genistein (G) and raloxifene (Ral) showed increased DNA synthesis and CK. Ral inhibited completely all stimulations except DPN and to some extent D. The ERα-specific antagonist methyl-piperidino-pyrazole (MPP) and the ERβ-specific antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a]pyrimidin-3-yl] phenol (PTHPP) inhibited DNA synthesis, CK and reactive oxygen species (ROS) formation induced by estrogens according to their receptors affinity. The LO inhibitor baicaleine inhibited only E2, DPN and G's effects. E2 and Ral unlike all other compounds had no effect on ERα mRNA expression, while ERβ mRNA expression was stimulated by all compounds. All compounds modulated the expression of 12LO and 15LO mRNA, except E2, PPT and Ral for 12LO, and 12- and 15-HETE productions and stimulated ROS formation which was inhibited by NADPH oxidase inhibitors diphenyleneiodonium chloride (DPI) and N-acetyl cysteine and the estrogen inhibitor ICI. DPI did not affect hormonal-induced DNA and CK. In conclusion, we provide evidence for the separation of mediation via ERα and ERβ pathways in the effects of estrogenic compounds on osteoblasts, but the role of LO/HETE/ROS is unclear.     

Genomic instability in interspecific cell hybrids III. Repression of Colcemid resistance in hybrids suggests preferential β-tubulin expression

A Colcemid-resistant Chinese hamster line with an altered form of β-tubulin was used in studies of the expression of spindle proteins in interspecific cell hybrids. Eight hybrids between this line, and a Colcemid-sensitive mouse cell line, were studied. The altered hamster β-tubulin was not expressed as an increased resistance to Colcemid in any hybrid. Since the complete hamster chromosome complement was represented among the hybrids, the absence of altered β-tubulin is not due to segregation of the mutant hamster β-tubulin gene. We suggest either that the hamster β-tubulin gene is repressed in hybrids, or that hamster β-tubulin is excluded from the spindle in hybrid cells. We compare these findings with previous reports of the repression of other highly active, moderately repeated constitutive genes in interspecific hybrids.     

Carbendazim-resistance associated β2-tubulin substitutions increase deoxynivalenol biosynthesis by reducing the interaction between β2-tubulin and IDH3 in Fusarium graminearum

Microtubule is a well-known structural protein participating in cell division, motility and vesicle traffic. In this study, we found that β₂-tubulin, one of the microtubule components, plays an important role in regulating secondary metabolite deoxynivalenol (DON) biosynthesis in Fusarium graminearum by interacting with isocitrate dehydrogenase subunit 3 (IDH3). We found IDH3 negatively regulate DON biosynthesis by reducing acetyl-CoA accumulation in F. graminearum and DON biosynthesis was stimulated by exogenous acetyl-CoA. In addition, the expression of IDH3 significantly decreased in the carbendazim-resistant mutant nt167 (Fgβ₂^F167Y). Furthermore, we found that carbendazim-resistance associated β₂-tubulin substitutions reducing the interaction intensity between β₂-tubulin and IDH3. Interestingly, we demonstrated that β₂-tubulin inhibitor carbendazim can disrupt the interaction between β₂-tubulin and IDH3. The decreased interaction intensity between β₂-tubulin and IDH3 resulted in the decreased expression of IDH3, which can cause the accumulation of acetyl-CoA, precursor of DON biosynthesis in F. graminearum. Thus, we revealed that carbendazim-resistance associated β₂-tubulin substitutions or carbendazim treatment increases DON biosynthesis by reducing the interaction between β₂-tubulin and IDH3 in F. graminearum. Taken together, the novel findings give the new perspectives of β₂-tubulin in regulating secondary metabolism in phytopathogenic fungi.     

Effects of decorin on the expression of α-smooth muscle actin in a human myofibroblast cell line

Myofibroblasts are metabolically and morphologically distinctive fibroblasts expressing α-smooth muscle actin (α-SMA), and their activation plays a key role in development of the fibrotic response. In an activated state, myofibroblasts cease to proliferate and start to synthesize large amounts of extracellular component proteins. The expression of α-SMA correlates with the activation of myofibroblasts. Decorin, a member of the small leucine-rich proteoglycan gene family, has been implicated in the negative control of cell proliferation primarily by upregulating the expression of p21, a potent inhibitor of cyclin-dependent kinase. In order to examine the effect of decorin on myofibroblast cell growth, we rendered a human lung myofibroblast cell line, MRC-5, quiescent by either cell–cell contact or serum starvation, and examined the relationship between decorin and α-SMA expression in these cells. The expression of decorin in cells made quiescent by serum starvation was lower than that in cells made quiescent by cell–cell contact. In contrast, the expression of α-SMA in cells made quiescent by cell–cell contact was lower than that in cells made quiescent by serum starvation. Furthermore, forced expression of decorin was accompanied by a suppression of α-SMA expression, whereas knocking down of decorin expression by RNA interference increased the expression of α-SMA.     

Effects of ascorbic acid and ��-carotene on HepG2 human hepatocellular carcinoma cell line

Recent studies have demonstrated that vegetable rich diets have protective effects on the occurrence and prognosis of various cancers. In addition to dietary intakes, ascorbic acid and β-carotene are also taken as supplements. The aim of this study was to assess effects of ascorbic acid, β-carotene and their combinations on human hepatocellular carcinoma cell line HepG2. Ascorbic acid and β-carotene were applied to cells as plasma peak concentrations (70 and 8 μM, respectively) and their half concentrations (35 and 4 μM, respectively) for 24 and 48 h. Genotoxic and cytotoxic effects of ascorbic acid and β-carotene were evaluated by alkali single cell gel electrophoresis (SCGE), acridine orange/ethidium bromide staining patterns of cells (apoptosis and necrosis) and lipid peroxidation (thiobarbituric acid reactive substances, TBARS). Results of the SCGE demonstrated that both ascorbic acid and β-carotene caused DNA damage on HepG2 which were also concordant to increased apoptosis and necrosis of cells. Increased TBARS values also demonstrated increased lipid peroxidation in these cells. Results of the present study demonstrates that when dietary intakes of ascorbic acid and β-carotene and their relevant achievable plasma level concentrations were considered, both ascorbic acid and β-carotene induce genotoxic and cytotoxic damage on HepG2 together with increased oxidative damage in contrast to their protective effect on healthy cells. This may be correlated to oxidative status and balance of ROS in hepatocellular carcinoma cells.     

TGF-β-stimulated aberrant expression of class III β-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells

The class III β-tubulin isotype (β_III) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III β-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology in pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-β (TGF-β) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-β on the aberrant expression of class III β-tubulin and the intracellular signaling pathway mediating these changes. TGF-β-induced aberrant expression and O-linked-β-N-acetylglucosamine (O-GlcNac) modification of class III β-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-β also stimulated phosphorylation of ERK. TGF-β-induced aberrant expression of class III β-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-β stimulated aberrant expression of class III β-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-β stimulation and provide useful information towards understanding the pathogenesis of proliferative vitreoretinal diseases.     

Steered molecular dynamics simulation of the binding of the β2 and β3 regions in domain-swapped human cystatin C dimer

The crystal structure of the human cystatin C (hCC) dimer revealed that a stable twofold-symmetric dimer was formed via 3D domain swapping. Domain swapping with the need for near-complete unfolding has been proposed as a possible route for amyloid fibril initiation. Thus, the interesting interactions that occur between the two molecules may be important for the further aggregation of the protein. In this work, we performed steered molecular dynamics (SMD) simulations to investigate the dissociation of the β2 and β3 strands in the hCC dimer. The energy changes observed during the SMD simulations showed that electrostatic interactions were the dominant interactions involved in stabilizing the two parts of the dimer during the early stages of SMD simulation, whereas van der Waals (VDW) interactions and electrostatic interactions were equally matched during the latter stages. Furthermore, our data indicated that the two parts of the dimer are stabilized by intermolecular hydrogen bonds among the residues Arg51 (β2), Gln48 (β2), Asp65 (β3), and Glu67 (β3), salt bridges among the residues Arg53 (β2), Arg51 (β2), and Asp65 (β3), and VDW interactions among the residues Gln48 (β2), Arg51 (β2), Glu67 (β3), Asp65 (β3), Phe63 (β3), and Asn61 (β3). The residues Gln48 (β2), Arg51 (β2), Asp65 (β3) and Glu67 (β3) appear to be crucial, as they play important roles in both electrostatic and VDW interactions. Thus, the present study determined the key residues involved in the stabilization of the domain-swapped dimer structure, and also provided molecular-level insights into the dissociation process of the hCC dimer.     

High expression of class III β-tubulin has no impact on functional cancer cell growth inhibition of a series of key vinblastine analogs

Clinical association studies have implicated high expression of class III β-tubulin as a predictive factor for lower response rates and reduced overall survival in patients receiving tubulin binding drugs, most notably the taxanes. Because of the implications, we examined a series of key vinblastine analogs that emerged from our studies in functional cell growth inhibition assays for their sensitivity to high expression of class III β-tubulin (human non-small cell lung cancer cell line A549 vs taxol-resistant A549-T24). Unlike taxol, vinblastine and a set of key analogs 3–10 did not exhibit any loss in sensitivity toward A549-T24. The results suggest that vinblastine and related analogs are not likely prone to resistance derived from high expression of class III β-tubulin unlike the taxanes. Most significant are the results with 4–6, a subset of 20′ amide vinblastine analogs. They match or exceed the potency of vinblastine and they display more potent activity against taxol-resistant A549-T24 than even wild type A549 cells (1.2–2-fold), complementing our prior observations that they also display no sensitivity to overexpression of Pgp (HCT116/VM46 vs HCT116) and are not subject to resistance derived from Pgp efflux.