Role In Metastatic Behavior Research Articles

Tumor-Intrinsic Galectin-3 Suppresses Melanoma Metastasis

Melanoma poses a poor prognosis with high mortality rates upon metastasis. Exploring molecular mechanisms governing melanoma progression paves the way for developing novel approaches to control melanoma metastasis and ultimately enhance patient survival rates. Extracellular galectin-3 (Gal-3) has emerged as a pleiotropic promoter of melanoma metastasis, exerting varying activities depending on its interacting partner. However, whether intracellular Gal-3 promotes melanoma aggressive behavior remains unknown. Here, we explored Gal-3 expression in human melanoma tissues as well as murine melanoma models to examine its causal role in metastatic behavior. We found that Gal-3 expression is downregulated in metastatic melanoma tissues compared to its levels in primary melanomas. Enforced silencing of Gal-3 in melanoma cells promoted migration, invasion, colony formation, in vivo xenograft growth and metastasis, and activated canonical oncogenic signaling pathways. Moreover, loss of Gal-3 in melanoma cells resulted in upregulated expression of the pro-metastatic transcription factor, nuclear factor of activated T cells (NFAT1), and its downstream metastasis-associated proteins, matrix metalloproteinase-3 (MMP-3) and interleukin-8 (IL-8). Overall, our findings implicate melanoma intracellular Gal-3 as a major determinant of its metastatic behavior and reveal a negative regulatory role for Gal-3 on the expression of NFAT1 in melanoma cells.

Mathematical Analysis of Two Competing Cancer Cell Migration Mechanisms Driven by Interstitial Fluid Flow

Recent experimental work has revealed that interstitial fluid flow can mobilize two types of tumor cell migration mechanisms. One is a chemotactic-driven mechanism where chemokine (chemical component) bounded to the extracellular matrix (ECM) is released and skewed in the flow direction. This leads to higher chemical concentrations downstream which the tumor cells can sense and migrate toward. The other is a mechanism where the flowing fluid imposes a stress on the tumor cells which triggers them to go in the upstream direction. Researchers have suggested that these two migration modes possibly can play a role in metastatic behavior, i.e., the process where tumor cells are able to break loose from the primary tumor and move to nearby lymphatic vessels. In Waldeland and Evje (J Biomech 81:22–35, 2018), a mathematical cell–fluid model was put forward based on a mixture theory formulation. It was demonstrated that the model was able to capture the main characteristics of the two competing migration mechanisms. The objective of the current work is to seek deeper insight into certain qualitative aspects of these competing mechanisms by means of mathematical methods. For that purpose, we propose a simpler version of the cell–fluid model mentioned above but such that the two competing migration mechanisms are retained. An initial cell distribution in a one-dimensional slab is exposed to a constant fluid flow from one end to the other, consistent with the experimental setup. Then, we explore by means of analytical estimates the long-time behavior of the two competing migration mechanisms for two different scenarios: (i) when the initial cell volume fraction is low and (ii) when the initial cell volume fraction is high. In particular, it is demonstrated in a strict mathematical sense that for a sufficiently low initial cell volume fraction, the downstream migration dominates in the sense that the solution converges to a downstream-dominated steady state as time elapses. On the other hand, with a sufficiently high initial cell volume fraction, the upstream migration mechanism is the stronger in the sense that the solution converges to an upstream-dominated steady state.

Abstract C020: Quantitative measurements of functional activity of the TGFβ and MAPK-AP1 pathways in colon cancer provides information on their role in cancer development and metastasis

Abstract Background: The advancement of targeted drugs for blocking tumor driving signaling pathway(s) is expected to lead to improved outcome for cancer patients. However, a critical requirement for that is reliable and accurate diagnostics on activity of oncogenic signaling pathways. A novel analysis method, based on Bayesian network-based inference of signaling pathway activity from levels of target gene mRNAs of the pathway-associated transcription factor, was developed to infer pathway activity from mRNA expression data [1], with which we previously reported that the WNT [1] and PI3K pathway [2] become active in colon carcinomas. Methods. Public datasets from the Gene Expression Omnibus, measured with the Affymetrix U133 Plus 2.0 platform, were analyzed for signaling pathway activity scores, among which are the MAPK-AP1, and TGFβ pathways. Colon tissue samples have been classified using the Consensus Molecular Subtypes of colorectal cancer classification (CMS). [3] Results. WNT and PI3K pathway activity were increased in colon carcinomas as reported before. The signaling pathway activity described by the CMS classification was confirmed using pathway activity analysis, TGFβ pathway activity was highest in the bad prognosis CMS4 subtype. A Kaplan-Meier curve shows higher TGFβ pathway activity to be associated with lower survival chance (p < 0.0001). Higher TGFβ pathway activity is seen in colon cancer metastases compared to primary tumors (p < 1e-13, based on 13 independent datasets). MAPK-AP1 pathway activity was increased (p < 1e-15, based on 18 analyzed datasets) in primary tumor samples compared to normal colon tissue, as demonstrated across multiple datasets, including a dataset with matched primary tumor and normal adjacent samples. Conclusion. MAPK-AP1 pathway activity in colon cancer is in line with frequent KRAS mutations that activate this pathway, while the dynamic range in activity may suggest a variation in sensitivity to targeted therapy. Results suggest that the lower survival associated with the CMS4 subtype is caused by increased TGFβ pathway activity, which is known to play a role in metastatic behavior, especially when activated in the presence of an active MAPK-AP1 signaling pathway, through interaction between the respective associated SMAD and c-Jun transcription factors [4]. Activity of these pathways can be measured simultaneously in a quantitative manner in an individual patient sample. They are clinically actionable and provide targets for drug-based therapy. Further clinical studies will be directed towards confirmation of the results and exploration of the clinical value of potential therapeutic approaches to prevent metastatic behavior in CRC on a personalized basis. References. [1] Cancer Res, vol. 74, no. 11, pp. 2936–2945, Jun. 2014. [2] The American Journal of Pathology, vol. 188, no. 9, pp. 1956–1972, Sep. 2018. [3] Nature Medicine, vol. 21, no. 11, pp. 1350–1356, Nov. 2015. [4] Cell, vol. 134, no. 2, pp. 215–230, Jul. 2008. Citation Format: Yvonne Wesseling-Rozendaal, Paul van Swinderen, Julie Gil, Sieglinde Neerken, Anja van de Stolpe. Quantitative measurements of functional activity of the TGFβ and MAPK-AP1 pathways in colon cancer provides information on their role in cancer development and metastasis [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C020. doi:10.1158/1535-7163.TARG-19-C020

Abstract 94: DDX31 cooperates with mutant p53 and EGFR to promote the multistep progression of invasive bladder cancer

Abstract Bladder cancer is the most common malignancy of the urinary tract worldwide. Approximately 70% of cases are diagnosed as non-muscle-invasive bladder cancer (NMIBC), while the remaining 30% of cases are classified as muscle-invasive bladder cancer (MIBC). Evidence based on molecular biology has highlighted that the aggressiveness of MIBC advances through a multistep mechanism due to many genomic alterations. Notably, alterations of TP53 and EGFR pathways frequently occur in bladder cancers and are associated with poor prognosis, respectively. However, the connection between the overexpression of EGFR and the p53 mutation in multistep carcinogenesis and the progression of MIBC remains unknown. Here we report the distinct critical roles of DEAD box polypeptide 31 (DDX31) in the multistep progression of muscle invasive bladder cancer (MIBC) through its sequential interaction with mutant p53 (mutp53) and EGFR. In early MIBC cells, nuclear DDX31 acts as a transcriptional co-activator that binds to mutp53/SP1 and enhances mutp53 transcriptional activation, thereby upregulating the EPB41L4B gene, which plays a critical role in metastatic behavior and promotes the invasion and migration of MIBC. Notably, in advanced MIBC, cytoplasmic DDX31, which is transported from the nucleus, functions as an adaptor scaffold protein that forms a complex with EGFR via its interaction with phosho-nucleolin (NCL), leading to constitutive activation of EGFR-Akt signaling. Significantly, high expression of both cytoplasmic DDX31 and p53 proteins is correlated with a poor prognosis in MIBC patients. More importantly, blocking the DDX31-NCL interaction via a dominant-negative peptide led to downregulation of EGFR-Akt signaling, resulting in a significant anti-tumoral effect of bladder cancer in vivo. Our findings reveal that DDX31 cooperates with mutp53 and EGFR to promote the multistep progression of MIBC and that inhibition of DDX31-NCL formation may lead to potential treatment strategies for advanced MIBC. Citation Format: Toyomasa Katagiri, Kei Daizumoto, Tetsuro Yoshimaru, Yosuke Matsushita, Tomoya Fukawa, Masaya Ono, Hiro-omi Kanayama. DDX31 cooperates with mutant p53 and EGFR to promote the multistep progression of invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 94.

Abstract 5115: The role of Cdc42-interacting protein 4 (CIP4) in osteosarcoma tumorigenesis

Abstract Osteosarcoma (OS) remains a devastating disease and requires better understanding of its biology for the development of novel therapeutic approaches. Tumor growth and metastases formation require an ability of cells to have a flexible cytoskeletal organization. Cdc42-interacting protein 4 (CIP4) is one of the scaffolding cytoskeleton-regulatory protein, whose role in tumors remains unknown. In the current study we investigated the role of CIP4 in OS tumorigenesis. We transfected highly aggressive mouse OS cells, DLM8 with the plasmid that downregulated expression of CIP4 protein (DLM8/shCIP4). We found that the growth rate of DLM8/shCIP4 clones was reduced by 30% when compared with non-transfected DLM8 cells or DLM8 cells transfected with control plasmid (DLM8/sh). When DLM8/shCIP4 cells were injected into mice subcutaneously they formed tumors that grew significantly slower than DLM8 or DLM8/sh control cells (p<0.02). We also found that inhibition of CIP4 expression in DLM8 cells decreased their motility and invasion properties in vitro, indicating on its ability to impair metastases growth. Fluorescent immunostaining for actin in DLM8/shCIP4 cells revealed lower perinuclear actin stress filaments formation and lower actin bridge to the lipid bilayer of the lipid cell membrane when compared with wild-type DLM8 or DLM8/sh. We also observed that downregulation of CIP4 changed cell membrane morphology in DLM8 cells - they had less protrusions and smoother cell surface than control cells. All these findings indicate that CIP4 may also play a role in metastatic behavior of OS cells and needs further investigation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5115.

Abstract 3166: Inspecting the presence of sialic acid α-2-3- galactose/GalNAC terminal sugar in human stomach adenocarcinoma by lectin histochemistry

Abstract Introduction: Glycosilation is the most frequent chemical change related to tumorogenesis which plays an important role in metastatic behavior of tumoral cells. Results of research have shown that changes in terminal sugar of glycoconjugates appear in the process of cancerization. Lectins are specific glycoproteins existing in plant and animal cells which specially attach to terminal sugar of glycoconjugates. This property of lectins make them significant markers in studies relating to histochemistry, biochemistry, recognition and differentiation of cancerous cells. The aim of this study is inspecting the presence of sialic acid α-2-3- galactose/GalNAC terminal sugar in different degrees of differentiation in human stomach adenocarcinoma by using MAA lectin in order to compare the amount of presenting this molecule in different stages and its relation with tumor progression and also for finding useful diagnostic and therapeutic method. Methods: Tissue samples of 40 stomach adenocarcinoma with different differentiation (well, moderately, poorly and undifferentiated) accompanying with normal tissues were selected from Pathology Department of Imam Reza Hospital in Mashhad, Iran and after studing H&E samples for assuring previous diagnosis, 5micrometer sections were prepared and were stained with MAA/Alcian blue pH 2.5 and then were considered microscopic inspection. Results: Microscopic study of samples showed no reaction in different degrees of stomach adenocarcinoma that indicates no presentence of sialic acid α-2-3- galactose/GalNAC terminal sugar, and the only positive reaction was between red blood cells of tissue vessels and MAA lectin. Conclusion: sialic acid α-2-3- galactose/GalNAC terminal sugar is not probably present in adult stomach tissue and is not produced in the changes that result from cancerization of cells, so it probably doesn't have an important and fundamental role in cellular interactions and in making special behaviors that result from neoplasic changes in gastric gland cells. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3166.

High aldehyde dehydrogenase and expression of cancer stem cell markers selects for breast cancer cells with enhanced malignant and metastatic ability

Cancer stem cells (CSCs) have recently been identified in leukaemia and solid tumours; however, the role of CSCs in metastasis remains poorly understood. This dearth of knowledge about CSCs and metastasis is due largely to technical challenges associated with the use of primary human cancer cells in pre-clinical models of metastasis. Therefore, the objective of this study was to develop suitable pre-clinical model systems for studying stem-like cells in breast cancer metastasis, and to test the hypothesis that stem-like cells play a key role in metastatic behaviour. We assessed four different human breast cancer cell lines (MDA-MB-435, MDA-MB-231, MDA-MB-468, MCF-7) for expression of prospective CSC markers CD44/CD24 and CD133, and for functional activity of aldehyde dehydrogenase (ALDH), an enzyme involved in stem cell self-protection. We then used fluorescence-activated cell sorting and functional assays to characterize differences in malignant/metastatic behaviour in vitro (proliferation, colony-forming ability, adhesion, migration, invasion) and in vivo (tumorigenicity and metastasis). Sub-populations of cells demonstrating stem-cell-like characteristics (high expression of CSC markers and/or high ALDH) were identified in all cell lines except MCF-7. When isolated and compared to ALDH(low)CD44(low/-) cells, ALDH(hi)CD44(+)CD24(-) (MDA-MB-231) and ALDH(hi)CD44(+)CD133(+) (MDA-MB-468) cells demonstrated increased growth (P < 0.05), colony formation (P < 0.05), adhesion (P < 0.001), migration (P < 0.001) and invasion (P < 0.001). Furthermore, following tail vein or mammary fat pad injection of NOD/SCID/IL2gamma receptor null mice, ALDH(hi)CD44(+)CD24(-) and ALDH(hi)CD44(+)CD133(+) cells showed enhanced tumorigenicity and metastasis relative to ALDH(low)CD44(low/-) cells (P < 0.05). These novel results suggest that stem-like ALDH(hi)CD44(+)CD24(-) and ALDH(hi)CD44(+)CD133(+) cells may be important mediators of breast cancer metastasis.

Inhibition of PAI-1 expression in breast cancer carcinoma cells by siRNA at nanomolar range

Plasminogen activator inhibitor type I (PAI-1) plays a central role in metastatic behavior by increasing cells' migratory capacities as shown in several tumoral cell lines. Moreover, in vivo high expression of this factor helps tumoral growth, both by its role in extracellular matrix remodeling and by favoring angiogenesis. High levels of PAI-1 are correlated with bad prognosis in several cancers, particularly in breast cancer. The effect of PAI-1 upon angiogenesis is also involved in atherosclerosis, in which high levels of PAI-1 expression are observed. Breast carcinoma MDA MB 231 cells are known for both having important metastatic capacities and expressing high levels of PAI-1. We have demonstrated in these cells that the transfection of PAI-1 specific small interfering RNAs (siRNA) specifically inhibited the expression of this factor by 91%. We evaluated siRNA activity by determining PAI-1 mRNA level, as well as intracellular and extracellular PAI-1 protein by using RT Q-PCR, Western blot and ELISA analyses, respectively. Data confirmed inhibition at mRNA levels (primary aim of interference), intracellular protein, and secreted PAI-1, the latter being operative successfully in the cell microenvironment. The lipidic vector Delivery Liposomes System (DLS) used was adapted to siRNA delivery as observed by particle size distribution analysis, confocal microscopy and transfection into MDA MB 231, in the presence of serum. SiRNA activity was clearly detected at concentrations as low as 10 nM. Moreover, the low cytotoxicity of this vector makes it a good candidate for future in vivo siRNA delivery.

Constitutive Activation of Wnt/β-Catenin Signaling Pathway in Migration-Active Melanoma Cells: Role of LEF-1 in Melanoma with Increased Metastatic Potential

A constitutive complex of β-catenin and LEF-1 has been detected in melanoma cell lines expressing either mutant β-catenin or mutant APC (Rubinfeld et al., Science, 275, 1790–1792, 1997). However, it has been recently reported that β-catenin mutations are rare in primary malignant melanoma, but its nuclear and/or cytoplasmic localization, a potential indicator of Wnt/β-catenin pathway activation, is frequently observed in melanoma (Rimm et al., Am. J. Pathol., 154, 325–329, 1999). In human malignant melanoma, the appearance of the tumorigenic phase represents a capacity for metastasis and is the significant phenotypic step in disease progression. Cell motility in invasive melanoma is thought to play a crucial role in metastatic behavior. In this work, we sought to determine which transcription factor of the LEF/TCF family was preferentially involved in human melanoma from different stages of tumor progression. We show that LEF-1 mRNA expression is predominant in highly migrating cells from metastatic melanomas. These actively migrating melanoma cells showed nuclear and cytoplasmic accumulation of β-catenin and active transcription from a reporter plasmid of the LEF/TCF binding site. These results may provide a new insight into the role of the Wnt/β-catenin signaling pathway in the tumor progression of malignant melanoma.

Activated K-ras is involved in regulation of integrin expression in human colon carcinoma cells.

Integrins participate in controlling proliferation and migration. Therefore, changes in integrin expression might be responsible for unrestrained proliferation and invasiveness of tumor cells. Alterations of integrin subunit expression have been observed in human colon carcinoma, especially loss or reduction of the alpha5 subunit, which was observed consistently. The mechanisms responsible for reduction of alpha5 expression and alteration of expression of other integrins are not fully understood. Circumstantial evidence from previous investigations points to an involvement of activated ras oncogenes in repression of integrin expression. The K-ras protooncogene is activated by point mutation in 50% of human colon carcinomas. Thus, we choose an antisense approach for specific inactivation of activated K-ras in the human colon carcinoma cell line SW 480 in order to test whether activated K-ras contributes to changes in integrin expression on colon carcinoma cells. Cell surface expression of the alpha1 and the alpha5 subunit was increased in K-ras antisense transfected clones, cell surface expression of the alpha3 subunit and the alphav subunit was decreased. This shows, in a human system, that activated K-ras is involved in diminishing cell surface expression of the alpha1beta1 collagen/laminin receptor and the alpha5beta1 fibronectin receptor, both of which are implicated in maintenance of a non-transformed phenotype. Moreover, activated K-ras contributes to increased cell surface expression of the alpha3beta1 laminin/collagen/fibronectin receptor and the alphavbeta5 vitronectin receptor, which might play a role in metastatic behavior of tumor cells.

Integrin α6β1 role in metastatic behavior of human pancreatic carcinoma cells

Integrin α6β1 role in metastatic behavior of human pancreatic carcinoma cells

Integrin alpha6beta1 role in metastatic behavior of human pancreatic carcinoma cells.

The factors that determine the metastatic behavior of pancreatic tumor cells are incompletely understood. In this study, we first demonstrate differences in adhesion properties, integrin expression and in vivo integrin function in the metastatic tumor cell line PaTu 8988s compared with the non-metastatic cell line PaTu 8988t. Both cell lines were derived from the same original tumor and exhibit identical genetic fingerprints. Using in vitro adhesion assays performed on purified extracellular matrix components, adhesion of PaTu 8988s cells was significantly increased on the basal membrane component laminin and decreased on the interstitial matrix protein fibronectin compared to PaTu 8988t cells. By immunocytochemistry and flow cytometry, and in correspondence with their adhesive properties, the metastatic PaTu 8988s cells did express a distinct pattern of integrin subunits. Laminin-binding integrins alpha6 and beta4 were overexpressed in PaTu 8988s cells. Fibronectin-binding alpha5 integrins were present at higher levels in the non-metastatic PaTu 8988t cells, whereas the beta1 subunit expression did not differ. Adhesion to laminin or fibronectin was specific and was mediated via integrins alpha6beta1 and alpha5beta1, respectively. In addition, metastasis formation in vivo after injection of cells into the tail vein of nude mice was inhibited by preincubation of PaTu 8988s cells with antibodies directed against the integrin alpha6 or beta1. We conclude that alpha6beta1 integrins are overexpressed and functionally active in metastatic human pancreatic carcinoma cells, and participate in metastasis formation probably through binding to the basal membrane component laminin.

Oncogene profile of papillary thyroid carcinoma

Background: Our purpose was to study the expression of multiple oncogenes in papillary thyroid cancer for possible interactions and prognostic significance. Methods: Twenty papillary thyroid carcinomas were studied for expression/mutation of 3 oncogenes: ras, ret/PTC, and erbB-2/neu. H, N, and K ras codons were examined by polymerase chain reaction (PCR), single-stranded conformation polymorphism, and sequencing. The thyroid oncogene ret/PTC was identified by reverse transcription (RT)-PCR. Gene amplification of erbB-2/neu was analyzed by differential PCR. The transmembrane domain of erbB-2/neu was sequenced for activating mutations. Quantitation of erbB-2/neu mRNA was evaluated by competitive RT-PCR, and protein expression was determined by immunohistochemistry. Results: Among 20 tumors, 3 had insular/anaplastic dedifferentiation, 13 were intrathyroidal, and 7 were metastatic to cervical lymph nodes (6) or lung (1). An H- ras 13 mutation was found in 1 metastatic tumor and an N- ras 61 mutation in 1 intrathyroidal tumor. ret/PTC was identified in 3 intrathyroidal and 5 metastatic tumors. No erbB-2/neu DNA amplification or mutations were identified, although 4 tumors had elevated erbB-2/neu mRNA levels. Three of 20 patients had abnormalities detected in multiple oncogenes; 2 had elevated erbB-2/neu mRNA and ret/PTC rearrangements, and 1 of these had pulmonary metastasis. An intrathyroidal papillary cancer had an N61 ras mutation and a ret/PTC gene rearrangement. Conclusions: ret/PTC rearrangements are present in 40% of papillary thyroid carcinomas and may play a role in metastatic behavior. In contrast, ras mutations are rare (10%). erbB-2/neu gene amplification and activating mutations are not detected, although elevated mRNA levels were found in 20% of papillary carcinomas. The lack of correlation among the 3 oncogenes in 17 of 20 (85%) papillary thyroid carcinomas suggests that they were not cumulative factors in the pathogenesis of these tumors. (Surgery 1999;125:46-52.)

The Association of the Nm23-M1 Protein and β-Tubulin Correlates with Cell Differentiation

The Nm23 protein is a nucleoside diphosphate kinase (NDPK) and is thought to play a critical role in metastatic behavior. It has been reported that a NDPK activity is present in microtubules assembled in vitro. Since microtubule assembly is determinant in cell growth and differentiation, we investigated whether Nm23-M1 forms molecular complexes with β-tubulin in murine cells either actively proliferating or differentiating. For this purpose a polyclonal antibody against the GST-Nm23-M1 fusion protein was generated and employed to detect Nm23-M1/β-tubulin complexes in murine tumor cells derived from the Lewis lung carcinoma (3LL) and in undifferentiated and differentiated myogenic cells (C2C12). Immunoblotting and immunoprecipitation experiments performed using the anti-fusion protein antibody demonstrated that the Nm23-M1 protein is detectable in in vitro tumor cell lines and in in vivo primary tumors but not in spontaneous lung metastases. These data are in good agreement with data previously reported. Immunoprecipitation experiments demonstrated that the Nm23-M1 protein forms complexes with β-tubulin in in vitro tumor cell lines, but not in primary tumors. Furthermore, the Nm23-M1 protein forms complexes with β-tubulin in myogenic cells prior to and after differentiation. Interestingly, however, the level of the Nm23-M1/β-tubulin complexes is remarkably increased in differentiated myotubes. In conclusion, the results indicate that the Nm23-M1 protein forms molecular complexes with β-tubulin and that the number of complexes increases during the differentiation process of murine cells.