Back to Old Books Toward Affordable Research: Homemade Phenol-Based Reagent for Triphasic RNA Purification.

Covid-19 crisis did hit many socio-economic aspects in the whole world. In the scientific research, the problem is getting even worse, since most of materials and consumable are allocated to the health sector. Many research laboratories around the world have big delay in receiving their purchases to accomplish their research projects. In the developing countries, the situation is much more difficult, since most of the funding resources are directed to the Covid-19 crisis and there is a notable increase in reagents' prices. Therefore, the aim of the present study is to make a homemade reagents for RNA purification from eukaryotic cells/tissues. The homemade phenol-based RNA extraction reagents were prepared using saturated phenol pH 4.3 (adjusted by 0.5 M citrate buffer) and guanidine thiocyanate. To validate the phenol-based reagent, RNA was purified from different biological samples (cell line, tissues, and fungi) using homemade phenol-based versus a commercial one. Concentration of RNA samples extracted from the same number of cells were compared to assess the homemade preparation of phenol-based reagent. In conclusion, homemade phenol-based reagent is cost effective and comparable to the commercial one. Using homemade phenol-based, RNA extraction was successfully purified from different biological sources.

PTEN mutations prevalence in HER2-positive breast cancer patients / Prevalencia de mutaciones en pten en pacientes con cáncer de mama HER2-positivo

Introduction: HER2-positive tumors is one of the aggressive subtypes of breast cancer that indicate bad prognosis. Trastuzumab is one of the targeted therapy which inhibit HER2 receptors. Mutations/expression deregulations of the downstream of HER2 receptors could cause resistance to trastuzumab. PTEN is a tumor suppressor gene which directly regulates PI3K pathway which renders it one of the predictive markers of trastuzumab. Methods: In the present study, PTEN mutations were screened in 51 patients with HER2-positive breast cancer. Also, 16 patients were further analyzed for protein expression. Results: The mutations were detected in 3 out of 51 patients (5.9%). In addition, 56.3% of the 16 patients showed downregulation/loss of PTEN protein expression. The loss of PTEN was found in 75% of estrogen-receptor negative patients (p =0.130). Conclusions: The downregulation/loss of PTEN protein has the tendency to be associated with ER-negative reflecting its value as a treatment prediction marker. (AU)

Introducción: Los tumores HER2 positivos son uno de los subtipos agresivos de cáncer de mama que indican un mal pronóstico. Trastuzumab es una de las terapias dirigidas que inhiben los receptores HER2. Las mutaciones/desregulaciones de la expresión aguas abajo de los receptores HER2 podrían causar resistencia a trastuzumab. PTEN es un gen supresor de tumores que regula directamente la vía PI3K, lo que lo convierte en uno de los marcadores predictivos de trastuzumab. Metodos: En el presente estudio, las mutaciones de PTEN se examinaron en cincuenta y un pacientes con cáncer de mama positivo para HER2. Además, dieciséis pacientes fueron analizados más a fondo para determinar la expresión de proteínas. Resultados: Las mutaciones se detectaron en tres de 51 pacientes (5.9%). Además, el 56,3 % de los dieciséis pacientes mostró regulación baja/pérdida de la expresión de la proteína PTEN. También se encontró pérdida de PTEN en el 75% de los pacientes con receptores de estrógeno negativos. Conclusiones: La regulación baja/pérdida de la proteína PTEN tiende a asociarse con ER-negativo, lo que refleja su valor como marcador de predicción de tratamiento. (AU)

Homemade plasmid Miniprep solutions for affordable research in low-fund laboratories.

As a consequence of Covid-19 pandemic, the basic lab consumables are in shortage, especially in the low-income countries. Thus, the main objective of the present study is to develop and evaluate homemade solution to isolate plasmid. To pursue this objective, RNase A was overexpressed in Bl21 DE3 cells (E. coli strain) and prepared as crude refolding reaction with proper activity. Also, lysis buffers, neutralization buffer, and washing buffers were prepared. The homemade miniprep kit showed successful isolation of the px48SpCas9 plasmid. The prepared plasmid purity was enough to be used successfully in PCR amplification. In addition, to get extra benefits from this study, seven primers were designed to match the plasmid backbone to produce DNA ladder (100-1500 bp). In conclusion, we were able to have attainable working solutions for plasmid miniprep and DNA ladder.

Assessment of galectins -1, -3, -4, -8, and -9 expression in ovarian carcinoma patients with clinical implications.

BACKGROUND AND AIM: Galectins have been recently tackled by many researchers in the field of cancer due to their role in tumorigenesis, disease progression, and metastasis. Thus, they are currently involved in biomarkers research on several types of cancer. In ovarian cancers, few studies were carried out to evaluate galectins expression profiling. Hence, our present study was executed to evaluate the mRNA expression of galectins -1, -3, -4, -8, and -9 in epithelial ovarian cancers. METHODS: Fifty-six tumor samples of ovarian carcinomas were analyzed for mRNA expression using qRT-PCR, and fold-changes were calculated in comparison to tissue samples of 26 women with normal ovaries. RESULTS: The results of the present paper emphasize the importance of galectins as predictors for targeted therapy. LGALS1, LGALS3, LGALS4, LGALS8, and LGALS9 were found to be mostly overexpressed in ovarian carcinoma patients with the following percentage: 78.6%, 92.9%, 66.1%, 87.5%, and 85.7% respectively. Moreover, galectins -3 and -9 were found to be significantly elevated with lymph node metastasis (p = 0.044 and p = 0.011). Also, upregulation of galectin-1 and -9 were statistically significant in stages IIB, IIC, and IIIB (p = 0.002) in FIGO staging. CA19.9 is positively correlated to galectin-4 expression (p = 0.039). CONCLUSION: Our findings strengthen the role of galectins in carcinogenesis, disease progression, and lymphnode metastasis in ovarian carcinomas. And since these galectins are mostly overexpressed, they could be promising markers for targeted therapy to reduce disease progression and metastasis process.

In-house protocol: spin-based viral RNA purification.

A worldwide shortage of molecular biology consumables is in surge. This includes filter tips, nucleic acid purification kits, polymerases, reverse-transcriptase, and different types of reagents which are included in viral diagnostic kits. In developing countries, the problem is even worse, since there is few capital enterprise to adopt this kind of industry. So, our aim is to develop a suitable, functional, comparable to commercial ones, and affordable in-house protocol to purify viral RNA. We sought some published and commercial RNA purification solutions to set-up an in-house protocol for viral RNA extraction. Solution was prepared accordingly. Also, LPA (linearized polyacrylamide) carrier was evaluated. The whole setting of in-house solutions with addition of LPA carrier was compared to QIAamp viral RNA minikit solutions. Our results showed that linearized polyacrylamide (LPA) carrier in homemade solutions is comparable to poly A carrier which is used in the most commercial kit. In addition, the whole setting of RNA purification solutions did achieve the purpose of viral RNA purification. Also, the result was confirmed using sputum of a Sars-Cov2 infected patient. Our experiments did end up with an affordable homemade solutions for viral RNA purification.

Galectins dysregulation: A way for cancer cells to invade and pervade.

Galectins are sticky molecules that bind to ß-galactoside. Their interactions render them essential players in many cellular processes. The imbalance of galectin expression was reported in many diseases. In cancer, galectins interact with the extracellular matrix, evade the immune system, and potentially have broad interactions with blood components. In the last ten years, since 2010, we did focus on galectin research in different cancer types. Our findings showed an interaction between cancer cells and erythrocytes via galectin-4. Moreover, we found that upregulation of galectins was associated with lymph node metastasis in ovarian cancers. Hence, with this, we shortly review some important aspects of galectins and their potential importance in more profound understanding of cancer progression and the field of cancer biomarkers.

Impact of MDM2 promoter SNP55 (rs2870820) on risk of endometrial and ovarian cancer.

BACKGROUND: While large GWAS analyses have not found convincing associations between MDM2 promoter SNP55 and gynaecological cancers, SNP55 is in linkage disequilibrium with two other functional SNPs in the same promoter, likely to obscure associations between single SNPs and cancer risk. Here, we assessed the impact of SNP55 on risk of endometrial and ovarian cancer, including sub-analyses stratified for other functional SNPs in the region. MATERIAL AND METHODS: Using a custom LightSNiP assay, we genotyped SNP55 in two large hospital-based cohorts of patients with ovarian (n = 1,332) and endometrial (n = 1,363) cancer and compared genotypes to healthy female controls (n = 1,858). RESULTS: Among individuals harbouring the SNP309TT genotype, the minor SNP55T-allele was associated with a reduced risk of endometrial (dominant model: OR = 0.63; CI = 0.45-0.88; p = 0.01). Regardless of the genotype in neighbouring SNPs, the SNP55T-allele was also associated with a reduced risk of endometrial cancer before 50 years of age (dominant model: OR = 0.56; CI = 0.34-0.90; p = 0.02). No association between SNP55 status and ovarian cancer risk was observed. CONCLUSIONS: MDM2 SNP55T-allele may correlate with reduced risk for endometrial cancer in a SNP309T-, but not SNP309G, context.

Epithelial to mesenchymal transition (EMT) is associated with attenuation of succinate dehydrogenase (SDH) in breast cancer through reduced expression of SDHC.

BACKGROUND: Epithelial to mesenchymal transition (EMT) is a well-characterized process of cell plasticity that may involve metabolic rewiring. In cancer, EMT is associated with malignant progression, tumor heterogeneity, and therapy resistance. In this study, we investigated the role of succinate dehydrogenase (SDH) as a potential key regulator of EMT. METHODS: Associations between SDH subunits and EMT were explored in gene expression data from breast cancer patient cohorts, followed by in-depth studies of SDH suppression as a potential mediator of EMT in cultured cells. RESULTS: We found an overall inverse association between EMT and the SDH subunit C (SDHC) when analyzing gene expression in breast tumors. This was particularly evident in carcinomas of basal-like molecular subtype compared to non-basal-like tumors, and a low SDHC expression level tended to have a prognostic impact in those patients. Studies in cultured cells revealed that EMT was induced by SDH inhibition through SDHC CRISPR/Cas9 knockdown or by the enzymatic inhibitor malonate. Conversely, overexpression of EMT-promoting transcription factors TWIST and SNAI2 caused decreased levels of SDHB and C and reduced rates of SDH-linked mitochondrial respiration. Cells overexpressing TWIST had reduced mitochondrial mass, and the organelles were thinner and more fragmented compared to controls. CONCLUSIONS: Our findings suggest that downregulation of SDHC promotes EMT and that this is accompanied by structural remodeling of the mitochondrial organelles. This may confer survival benefits upon exposure to hostile microenvironment including oxidative stress and hypoxia during cancer progression.

The Synonymous Isocitrate Dehydrogenase 1 315C>T SNP Confers an Adverse Prognosis in Egyptian Adult Patients with NPM1-/CEBPA-Negative Acute Myeloid Leukemia.

Although the clinical features of isocitrate dehydrogenase (IDH) genetic aberrations have been well-characterized in acute myeloid leukemia (AML), definitive information on their prognostic significance is lacking. We aimed to explore the prognostic significance of IDH gene alterations in an Egyptian cohort of adult patients with de novo AML. Diagnostic peripheral blood samples from 51 AML patients were analyzed for the presence of mutations/SNPs in exon 4 of IDH1 and IDH2 genes using polymerase chain reaction amplification followed by direct sequencing. IDH mutational status had no impact on event-free survival (EFS) and overall survival (OS), whereas the presence of IDH1 315C>T SNP was significantly associated with inferior EFS (P = 0.037) and OS (P = 0.034) as compared with wild-type IDH1. IDH1 315C>T SNP but not IDH mutations is associated with unfavorable outcomes, suggesting that AML patients with IDH1 315C>T SNP can represent a new subgroup of patients which allows refined risk stratification.

Tumor cells interact with red blood cells via galectin-4 - a short report.

BACKGROUND: The ability of tumor cells to invade and metastasize is relevant to the process of cancer progression and, as such, it represents an obstacle to cancer cure. So far, limited information is available on interactions between circulating tumor cells and blood cells. It is well-documented that galectin-4 is upregulated in many types of tumor cells and is involved in metastasis. Here, we address the hypothesis that tumor cells may interact with red blood cells (RBCs) via galectin-4. METHODS: High galectin-4 expressing colon, normal pancreatic and pancreatic cancer-derived cell lines (n = 5) were incubated with peripheral blood cells from different donors. Their interactions and associated proteins were examined by immunostaining and live cell imaging. RESULTS: We found that (endogenous or exogenous) galectin-4 expressing tumor cells interact directly with RBCs. We also observed an accumulation of galectin-4 and human blood group antigens at the contact sites between these cells. By comparing the number of RBCs attaching to each tumor cell, we found that cells with high pre-incubation expression levels of galectin-4 attached significantly more RBCs than those with low expression levels (p < 1 × 10-7). Conversely, we found that RBC attachment induces galectin-4 expression in tumor cells. CONCLUSIONS: From our data we conclude that tumor cells directly interact with red blood cells via galectin-4.

PIK3CA mutations in HER2-positive Breast Cancer Patients; Frequency and Clinicopathological Perspective in Egyptian Patients

Missense mutations in PIK3CA are common in breast cancers. They mostly involve exons 9 and 20 which encode kinase and helical domains of the protein and may result in its activation. PIK3CA activating mutations were previously shown to predict lower pathologic complete response (pCR) in HER2-positive breast cancer cases undergoing neoadjuvant human epidermal growth factor receptor 2-targeting therapy. Hence, the present work was conducted to estimate the mutation frequency in PIK3CA in 51 HER2-positive patients by direct sequencing. Our results showed 8 out of 51 (15.7%) to harbor PIK3CA mutations in either exon 9 or 20, or both. Three patients had mutations in both exons 9 and 20. Seven (13.7%) possess missense mutations in exon 20 which changed the amino acid sequence of the protein (H1047R, M1040I, and G1049G). Only four cases harbored mutations in exon 9, changing the codon sequences (E545K E545A, and R524K). Taking the clinicopathological data to account, the mutation frequency was greater in ductal than lobular carcinomas, in grade II rather than III and in lymph node positive lesions, with a higher HER2 score and which are ER/PR negative. However, none of the correlations proved statistically significant. In conclusion, to the best of our knowledge, the PIK3CA mutation frequency in this study is the first report regarding HER2-positive breast cancer patients in Egypt. Hereby, we highlight a moderate frequency which could be useful in the future as a predictive marker for anti-HER2 therapy.

MDM2 promoter SNP55 (rs2870820) affects risk of colon cancer but not breast-, lung-, or prostate cancer.

Two functional SNPs (SNP285G > C; rs117039649 and SNP309T > G; rs2279744) have previously been reported to modulate Sp1 transcription factor binding to the promoter of the proto-oncogene MDM2, and to influence cancer risk. Recently, a third SNP (SNP55C > T; rs2870820) was also reported to affect Sp1 binding and MDM2 transcription. In this large population based case-control study, we genotyped MDM2 SNP55 in 10,779 Caucasian individuals, previously genotyped for SNP309 and SNP285, including cases of colon (n = 1,524), lung (n = 1,323), breast (n = 1,709) and prostate cancer (n = 2,488) and 3,735 non-cancer controls, as well as 299 healthy African-Americans. Applying the dominant model, we found an elevated risk of colon cancer among individuals harbouring SNP55TT/CT genotypes compared to the SNP55CC genotype (OR = 1.15; 95% CI = 1.01-1.30). The risk was found to be highest for left-sided colon cancer (OR = 1.21; 95% CI = 1.00-1.45) and among females (OR = 1.32; 95% CI = 1.01-1.74). Assessing combined genotypes, we found the highest risk of colon cancer among individuals harbouring the SNP55TT or CT together with the SNP309TG genotype (OR = 1.21; 95% CI = 1.00-1.46). Supporting the conclusions from the risk estimates, we found colon cancer cases carrying the SNP55TT/CT genotypes to be diagnosed at younger age as compared to SNP55CC (p = 0.053), in particular among patients carrying the SNP309TG/TT genotypes (p = 0.009).

The Liver MicroRNA Expression Profiles Associated With Chronic Hepatitis C Virus (HCV) Genotype-4 Infection: A Preliminary Study.

BACKGROUND: MicroRNAs (miRNAs) have been repeatedly shown to play important roles in liver pathologies, including hepatitis, liver cirrhosis, and liver cancer. Egypt has the highest hepatitis C virus (HCV) infection rate worldwide, predominantly involving genotype-4. OBJECTIVES: In this study, we attempted to characterize the miRNA profile of the poorly studied genotype 4 of HCV in chronically infected Egyptian patients to obtain a better understanding of the disease and its complications and help in the design of better management protocols. PATIENTS AND METHODS: We analyzed the expression levels of a selected panel of 94 miRNAs in fresh liver biopsies collected from 50 Egyptian patients diagnosed with chronic HCV infection using quantitative real-time polymerase chain reaction (PCR) assay. Non-parametric tests were used to analyze the expression level of each miRNA and association with the clinicopathological features of enrolled patients in this study. RESULTS: Our results revealed differential expression levels of the analyzed miRNAs compared to the normal controls. Twenty-seven miRNAs (including miR-105, miR-147, miR-149-3p, and miR-196b) showed up-regulation, while 17 miRNAs (including miR-21, miR-122, miR-199a-3p, and miR-223) showed down-regulation. An inverse correlation was observed between levels of miR-95, miR-130a, and miR-142-5p with the blood albumin level. Increased expression levels of seven miRNAs (miR-29c, miR-30c, miR-126, miR-145, miR-199a, miR-199a-3p, and miR-222) were observed with severe chronic hepatic inflammation. Several deregulated miRNAs found in this study have been previously linked to chronic liver inflammation and the risk of hepatocellular carcinoma (HCC) development. CONCLUSIONS: The identified expression profiles of some examined miRNAs might offer important points to consider for the treatment of naive patients and the management of chronically infected HCV patients in Egypt and around the world.

The tyrosine kinase inhibitor, sunitinib malate, induces cognitive impairment in vivo via dysregulating VEGFR signaling, apoptotic and autophagic machineries.

Chemobrain refers to a cluster of cognitive deficits which affects almost 4-75% of chemotherapy-treated cancer patients. Sunitinib, an FDA-approved multityrosine kinase inhibitor, is currently used in treating different types of tumors. Despite being regarded as targeted therapy which blunts sustained angiogenesis in cancer milieu through inhibiting vascular endothelial growth factor receptor 2 (VEGFR2) signaling, the latter has a cardinal role in cognition. Recent clinical reports warned that sunitinib adversely affected memory processing in cancer patients. Nevertheless, the underlying mechanisms have not been investigated yet. Hence, we explored the impact of a clinically relevant dose of sunitinib on memory processing in vivo and questioned the implication of VEGFR2 signaling, autophagy and apoptosis. Strikingly, sunitinib preferentially impaired spatial cognition as evidenced in Morris water maze, T-maze and passive avoidance task. Consistently, sunitinib degenerated cortical and hippocampal neurons as assessed by histopathological examination and toluidine blue staining. Ultrastructural examination also depicted chromatin condensation, mitochondrial damage and accumulated autophagosomes. Digging deeper, central VEGF/VEGFR2/mTOR signaling was robustly suppressed. Besides, sunitinib boosted cortical and hippocampal p53 and executioner caspase-3 and decreased nuclear factor kappa B and Bcl-2 levels promoting apoptotic cell death. It also profoundly impeded neuronal autophagic flux as shown by decreased beclin-1 and Atg5 and increased p62/SQTSM1 levels. To our knowledge, this is the first study to provide molecular insights into sunitinib-induced chemofog where impeded VEGFR2 signaling and autophagic and hyperactivated apoptotic machineries act in neurodegenerative concert. Importantly, our findings shed light on potential therapeutic strategies to be exploited in the management of sunitinib-induced chemobrain.

Promoter SNPs rs116896264 and rs73933062 form a distinct haplotype and are associated with galectin-4 overexpression in colorectal cancer.

Galectin-4 is a member of the galectin family which consists of 15 galactoside-binding proteins. Previously, galectin-4 has been shown to have a role in cancer progression and metastasis and it is found upregulated in many solid tumours, including colorectal cancer (CRC). Recently, the role in the metastatic process was suggested to be via promoting cancer cells to adhere to blood vascular endothelium. In the present study, the regulatory region of LGALS4 (galectin-4) in seven colon cell lines was investigated with respect to genetic variation that could be linked to expression levels and therefore a tumourigenic effect. Interestingly, qRT-PCR and sequencing results revealed that galectin-4 upregulation is associated with SNPs rs116896264 and rs73933062. By use of luciferase reporter- and pull-down assays, we confirmed the association between the gene upregulation and the two SNPs. Also, using pull-down assay followed by mass spectrometry, we found that the presence rs116896264 and rs73933062 is changing transcription factors binding sites. In order to assess the frequencies of the two SNPs among colon cancer patients and healthy individuals, we genotyped 75 colon cancer patients, 12 patients with adenomatous polyposis and 17 patients with ulcerative colitis and we performed data mining in the 1000 genomes databank. We found the two SNPs co-occuring in 21% of 75 CRC patients, 0 out of 12 patients of adenomatous polyposis, and 6 out of 17 patients (35%) with ulcerative colitis. Both in the patient samples and in the 1000 genomes project, the two SNPs were found to co-occur whenever present (D' = 1).

Induction of P3NS1 Myeloma Cell Death and Cell Cycle Arrest by Simvastatin and/or γ-Radiation.

The present study was conducted to investigate the effect of γ-radiation alone or combined with a cytotoxic drug, simvastatin, on viability and cell cycling of a myeloma cell line. P3NS1 myeloma cells were treated with the selected dose of simvastatin (0.1 µM/l) 24 hours prior to γ-irradiation (0.25, 0.5 and 1 Gy). The cell viability, induction of apoptosis, cell death, cell cycling, generation of ROS, and expression of P53, Bax, Bcl2, caspase3, PARP1 and Fas genes were estimated. The results indicated that simvastatin (0.1 µM/l) treatment for 24 hours prior to γ- irradiation increased cell death to 37.5% as compared to 4.81% by radiation (0.5 Gy) alone. It was found that simvastatin treatment before irradiation caused arrest of cells in G0/G1 and G2/M phases as assessed using flow cytometry. Interestingly, simvastatin treatment of P3NS1 cells increased the intracellular ROS production and decreased antioxidant enzyme activity with increased P53, Bax and Caspase3 gene expression while that of Bcl2 was decreased. Consequently, our results indicated that pre-treatment with simvastatin increased radio sensitivity of myeloma tumor cells in addition to apoptotic effects through an intrinsic mitochondrial pathway.

Expression profiling of cancer-related galectins in acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common type of leukemia in adults with the lowest survival rate of all the leukemias. It is a heterogeneous disease in which a variety of cytogenetic and molecular alterations have been identified. Some galectins were previously reported to have important roles in cancer-like neoplastic transformation, tumor cell survival, angiogenesis, and tumor metastasis. Previous studies have showed that some galectin family members play a role in various types of leukemia. The present study aims at evaluating and clarifying the diagnostic and prognostic value of the expression of cancer-related galectins in relation to the clinicopathological characters of AML patients. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect expression profile of eight galectin family members (galectin-1, -2, -3, -4, -8, -9, -12, and -13) in 53 newly diagnosed de novo AML patients. The samples were collected from the inpatient clinic at National Cancer Institute (NCI), Cairo University (CU), diagnosed between July 2012 and May 2013. Our results show that patients with lower LGALS12 gene expression have a lower overall survival than those with higher expression (P value <0.026). Moreover, a statistically significant association between the LGALS4 gene expression and patient age is found. Hence, the higher expression of LGALS4 gene is associated with younger age (adjusted P value <0.001). In conclusion, galectin-12 may be a potential prognostic marker for AML.

Analysis of DNA-protein interactions: from nitrocellulose filter binding assays to microarray studies.

Determination of the sequence of the human genome and knowledge of the genetic code have allowed rapid progress in the identification of mammalian proteins. However, far less is known about the molecular mechanisms that control expression of human genes and about the variations in gene expression that underlie many pathological states, including cancer. This is caused in part by lack of information about the binding specificities of DNA-binding proteins and particularly regulative important molecules such as transcription factors. It is consequently crucial to develop new technologies or improve existing ones for the analysis of DNA-protein interaction in order to identify and characterise DNA response elements and the related transcription factors or other DNA-binding proteins. The techniques that are currently available vary with respect to the type of result that can be expected from the assay: a mere qualitative demonstration of binding; the identification of response element sequences at high throughput; or a quantitative characterisation of affinities. This article gives an overview of early and recent methodologies applied to such ends.

Transcriptional activation of the beta-catenin gene at the invasion front of colorectal liver metastases.

beta-Catenin is a pivotal molecule of the Wnt-signalling pathway, involved in regulation of developmental and oncogenic processes as well as in intercellular adhesion. So far, beta-catenin has been thought to be regulated mainly at the protein level. Here, we provide evidence for a transcriptional mechanism of beta-catenin regulation at the invasion front of colorectal liver metastases. In a nude mouse/LS174T cell xenograft model of colorectal liver metastases, we observed beta-catenin up-regulation at the mRNA and protein levels and a 13.7-fold increase of beta-catenin promoter activity in the cancer cells of the invasion front. In addition, the promoter activity was five-fold higher in the interior of the tumour than in cells growing in cell culture. In vitro studies revealed binding of TCF-4 to the beta-catenin promoter and reduced promoter activity by over-expression of dominant negative TCF-4, or beta-catenin knock-down and increased activity by beta-catenin over-expression, indicating that beta-catenin acts as co-transcription factor of its own promoter. In 55% (7/13) of clinical specimens, beta-catenin mRNA was markedly elevated in the cancer cells of the invasion front. Elevation of mRNA was paralleled by increased nuclear and cytoplasmic beta-catenin protein concentrations. These data indicate that transcriptional regulation contributes to the dynamic changes of beta-catenin levels upon the confrontation of tumour cells with the host microenvironment.