Phenytoin and gingival mucosa: A molecular investigation.

Several distinct classes of drugs, such as anticonvulsants, immunosuppressants, and calcium channel blockers, caused gingival overgrowth. One of the main drugs associated with the gingival overgrowth is the anti-epileptic such as phenytoin, which affects gingival tissues by altering extracellular matrix metabolism. In our study, we evaluate the effect of phenytoin, a drug whose active substance is phenytoin, on gingival fibroblasts of healthy volunteers. Gene expression of 29 genes was investigated in gingival fibroblasts' cell culture treated with phenytoin compared with untreated cells. Among the studied genes, only 13 genes (CXCL5, CXCL10, CCR1, CCR3, CCR5, CCR6, IL-1A, IL-1B, IL-5, IL-7, IL-6R, BMP-2, and TNFSF-10) were statistically significant. All but one gene resulted downregulated after 24 h of treatment with phenytoin. BPM2 was the only, although weakly, up-expressed gene. Probably, we have not highlighted overexpression of the other inflammatory molecules because the study was performed on healthy people. Many studies show that phenytoin induces the overexpression of these cytokines but, probably, in our study, the drug does not have the same effect because we used gingival fibroblasts of healthy people.

Possible effect of SNAIL family transcriptional repressor 1 polymorphisms in non-syndromic cleft lip with or without cleft palate.

OBJECTIVE: Orofacial development is a complex process subjected to failure impairing. Indeed, the cleft of the lip and/or of the palate is among the most frequent inborn malformations. The JARID2 gene has been suggested to be involved in non-syndromic cleft lip with or without cleft palate (nsCL/P) etiology. JARID2 interacts with the polycomb repressive complex 2 (PRC2) in regulating the expression patterns of developmental genes by modifying the chromatin state. MATERIALS AND METHODS: Genes coding for the PRC2 components, as well as other genes active in cell differentiation and embryonic development, were selected for a family-based association study to verify their involvement in nsCL/P. A total of 632 families from Italy and Asia participated to the study. RESULTS: Evidence of allelic association was found with polymorphisms of SNAI1; in particular, the rs16995010-G allele was undertransmitted to the nsCL/P cases [P = 0.004, odds ratio = 0.69 (95% C.I. 0.54-0.89)]. However, the adjusted significance value corrected for all the performed tests was P = 0.051. CONCLUSIONS: The findings emerging by the present study suggest for the first time an involvement of SNAI1 in the nsCL/P onset. CLINICAL RELEVANCE: Interestingly, SNAI1 is known to promote epithelial to mesenchymal transition by repressing E-cadherin expression, but it needs an intact PRC2 to act this function. Alterations of this process could contribute to the complex etiology of nsCL/P.

Role of the MIR146A polymorphism in the origin and progression of oral squamous cell carcinoma.

Gene expression and cell behavior are regulated by several factors, including small non-coding RNAs. MicroRNAs affecting cell growth, differentiation, and apoptosis are thought to play an important role in tumorigenesis. The levels of miR-146 appear to be associated with cancer development and progression, including that of oral squamous cell carcinoma. The aim of this investigation was to ascertain whether the single nucleotide polymorphism, rs2910164, mapping in the MIR146A gene, has a role in oral squamous cell carcinoma progression. A genetic association study was performed with a sample set of 346 oral squamous cell carcinomas collected in Italy. Our data indicate that the rs2910164 polymorphism is not associated with tumor development. However, a slight increase in the frequency of the variant allele was observed in Stage II tumors. Further investigations are needed to verify a possible role of the variant allele or rs2910164 in oral squamous cell carcinoma progression.

No evidence of HAND2 involvement in nonsyndromic cleft lip with or without cleft palate.

Craniofacial morphogenesis is determined by multistep processes involving signalling molecules and transcription factors, which are organised into highly coordinated pathways. Derailment from this intricate network can lead to congenital malformations. Cells migrate from neural crests to populate different structures, such as branchial arches, involved in embryonal orofacial development. The EDN1 pathway is involved in branchial arch development. Gene knockout and knockdown experiments on EDN1 or its downstream effector dHAND resulted in mice that were characterised by craniofacial defects and cleft palate. Our aim was to evaluate whether the transcription factor HAND2 could be implicated in non-syndromic cleft lip with or without cleft palate (CL/P) aetiology. A sample study composed of 39 multiplex Italian pedigrees was enrolled to test linkage between two microsatellite flanking HAND2 locus and CL/P. No evidence of linkage between HAND2 and CL/P was obtained. Indeed, formal levels of exclusion were obtained with different inheritance models. Investigation results did not support a role of HAND2 in CL/P aetiology. Nevertheless a minor contribute of the gene in clefting could not be ruled out.

Evaluation of gene expression in MG63 human osteoblastlike cells exposed to tantalum powder by microarray technology.

Conventional orthopedic implants are composed from titanium. To improve some characteristics (ie, volumetric porosity, modulus of elasticity, frictional modulus), a new porous tantalum biomaterial has been developed and its biocompatibility reported. By using DNA microarrays containing 20,000 genes, several genes whose expression were significantly up- or down-regulated were identified in an osteoblastlike cell line (MG63) cultured with tantalum powder (TP). The differentially expressed genes cover a broad range of functional activities: signaling transduction; transcription; cell cycle regulation, proliferation, and apoptosis; and cytoskeleton formation. To the authors' knowledge, the data reported represent the first genetic portrait of TP.

New evidence for the role of cystathionine beta-synthase in non-syndromic cleft lip with or without cleft palate.

Orofacial clefts have a multifactorial aetiology encompassing both genetic and environmental components. While there is wide agreement on the importance of both genetic and nutritional factors, genetic influence in particular has not been well defined. As genetic variants in folate and homocysteine metabolism have been reported to influence the risk of orofacial clefts, an Italian cleft lip with or without cleft palate (CL/P) data set was enrolled for an analysis based on family association to test betaine-homocysteine methyltransferase (BHMT and BHMT2) and cystathionine beta-synthase (CBS) variants. No significant level of association was found between BHMT and BHMT2 variants, while evidence of an allelic association with CL/P was found for the single nucleotide polymorphism rs4920037, mapping at the CBS gene. A log-linear approach indicated that the best genetic model takes into account both mother and child genotypes. This suggests that human orofacial development is influenced by CBS genotypes that possibly operate through intergenerational fetal-maternal interaction.

Diazepam effects on non-syndromic cleft lip with or without palate: epidemiological studies, clinical findings, genes and extracellular matrix.

IMPORTANCE OF THE FIELD: This review analyses international studies investigating the combined genetic and environmental causes of cleft lip with or without cleft palate (CL/P) and describes successes and limitations in identifying underlying genetic and environmental factors. CL/P, the most common congenital facial malformation, is a major public health burden in terms of medical costs and emotional stress to patients and families. Because genetic and environmental factors determine risk of occurrence, CL/P has a complex, multifactor aetiology. AREAS COVERED IN THIS REVIEW: English language reports from 1980 to 2010 were searched for in Medline, PubMed, Science Citation Index, textbooks and review articles on drugs and pregnancy. Key words were diazepam or benzodiazepine(s) combined with cleft lip, cleft palate, oral malformations, prenatal exposure, GABA, gene expression and extracellular matrix. WHAT THE READER WILL GAIN: This review presents an updated assessment of the mutagenic and genotoxic effects of diazepam (DZ), one of the most commonly used benzodiazepines, on CL/P occurrence. TAKE HOME MESSAGE: Data are divergent; more studies are needed for an in-depth picture of the effects of DZ during gestation on the child's development, particularly on orofacial clefts.

New insights in collagen turnover in orofacial cleft patients.

OBJECTIVE: We aimed to characterize the fibroblast phenotype of patients by analyzing gene and protein expression of cleft lip and/or cleft palate fibroblasts in relation to collagen turnover and extracellular matrix remodeling. PATIENTS: Human palatal fibroblasts were obtained from three healthy subjects without cleft lip and/or cleft palate and from three subjects with nonsyndromic cleft lip and/or cleft palate. Collagen turnover-related gene and protein expression were analyzed by real-time polymerase chain reaction, Western and dot blots, and sodium dodecyl sulfate zymography. RESULTS: Cleft lip and/or cleft palate fibroblasts, compared with controls, displayed a down-regulation of collagens type I and III messenger RNA (p < .0001 and p < .001, respectively) but an opposite tendency to increase protein levels. Cleft lip and/or cleft palate cells had higher lysyl hydroxylase-2b messenger RNA levels expressed in relation to collagen type I messenger RNA, down-regulated matrix metalloproteinase-1, tissue inhibitor of matrix metalloproteinase-1, and Secreted Protein Acidic and Rich in Cysteine messenger RNA (p < .0001 and p < .01, respectively). Pro-matrix metalloproteinase-1 tended to decrease, and pro-matrix metalloproteinase-2 and -9 were down-regulated (p < .01, p < .05, respectively), as was Secreted Protein Acidic and Rich in Cysteine protein expression (p < .05). CONCLUSIONS: Our results suggest that the cleft lip and/or cleft palate fibroblast phenotype is characterized by a tendency toward interstitial collagen deposition due to posttranslational modifications, such as decreased collagen degradation by matrix metalloproteinases and increased collagen cross-links. These findings may contribute to the knowledge of the cleft lip and/or cleft palate fibroblast phenotype and may be useful to the surgeon when considering the potential wound contraction and subsequent undesired scarring in cleft lip and/or cleft palate ocurring after the surgical closure of a cleft palate.

Expression and association data strongly support JARID2 involvement in nonsyndromic cleft lip with or without cleft palate.

Nonsyndromic cleft lip with or without cleft palate (CL/P) affects approximately 1 in 1,000 births. Genetic studies have provided evidence for the role of several genes and candidate loci in clefting; however, conflicting results have frequently been obtained and much have to be done to unravel the complex genetics of CL/P. In the present investigation we have focused on the candidate region in 6p23, a region that have been found linked to CL/P in several investigations, in the attempt to find out the susceptibility gene provisionally named OFC1. Gene expression experiments in mice embryo of positional candidate genes revealed that JARID2 was highly and specifically expressed in epithelial cells in merging palatal shelves. A family-based linkage disequilibrium study confirmed the pivotal role of JARID2 in orofacial development and strongly supports a role for this gene in CL/P etiology (multiallelic haplotype test P=6 x 10(-5)). Understanding the molecular role of JARID2 within facial development may offer additional information to further unravel the complex genetics of CL/P.

Effects of pulsed electromagnetic fields on human osteoblastlike cells (MG-63): a pilot study.

BACKGROUND: Although pulsed electromagnetic fields (PEMFs) are used to treat delayed unions and nonunions, their mechanisms of action are not completely clear. However, PEMFs are known to affect the expression of certain genes. QUESTIONS/PURPOSES: We asked (1) whether PEMFs affect gene expression in human osteoblastlike cells (MG63) in vitro, and (2) whether and to what extent stimulation by PEMFs induce cell proliferation and differentiation in MG-63 cultures. METHODS: We cultured two groups of MG63 cells. One group was treated with PEMFs for 18 hours whereas the second was maintained in the same culture condition without PEMFs (control). Gene expression was evaluated throughout cDNA microarray analysis containing 19,000 genes spanning a substantial fraction of the human genome. RESULTS: PEMFs induced the upregulation of important genes related to bone formation (HOXA10, AKT1), genes at the transductional level (CALM1, P2RX7), genes for cytoskeletal components (FN1, VCL), and collagenous (COL1A2) and noncollagenous (SPARC) matrix components. However, PEMF induced downregulation of genes related to the degradation of extracellular matrix (MMP-11, DUSP4). CONCLUSIONS AND CLINICAL RELEVANCE: PEMFs appear to induce cell proliferation and differentiation. Furthermore, PEMFs promote extracellular matrix production and mineralization while decreasing matrix degradation and absorption. Our data suggest specific mechanisms of the observed clinical effect of PEMFs, and thus specific approaches for use in regenerative medicine.

Anorganic bovine bone (Bio-Oss) regulates miRNA of osteoblast-like cells.

Bio-Oss (Geistlich) is composed of an organic bovine bone and has been widely used in several bone regeneration procedures during oral surgery. However, how this biomaterial enhances osteoblast activity to promote bone formation is not completely understood. MicroRNAs (miRNAs) represent a class of small, functional, noncoding RNAs of 19 to 23 nucleotides that regulate the transcription of messenger RNAs (mRNAs) in proteins. In this study, the miRNA microarray technique was used to investigate translation regulation in an osteoblast-like cell line (MG63) exposed to Bio-Oss. Nine up-regulated miRNAs (mir-423, mir-492, mir-191, mir-23a, mir-377, mir-494, mir-214, mir-193b, mir-320) and 4 down-regulated miRNAs (mir-27a, mir-24, mir-188, let-7c) were identified. Because each miRNA regulates 100 mRNAs, only mRNAs related to bone formation were analyzed. The vast majority of detected mRNAs are down-regulated, including some homeobox genes (genes that regulate the morphogenesis of an entire segment of the body), such as noggin and EN1. An indirect positive effect was demonstrated on bone morphogenetic protein-4. To the authors' knowledge, the data reported here are the first on translation regulation in osteoblasts exposed to Bio-Oss. This study may be relevant in better understanding the molecular mechanism of bone regeneration and used as a potential tool for analyzing the combined use of cytokines.

PerioGlas® Acts on Human Stem Cells Isolated from Peripheral Blood.

BACKGROUND: PerioGlas® (PG) is an alloplastic material used for grafting periodontal osseous defects since 1995. In animal models, it has been proven that PG achieves histologically good repair of sur-gically created defects. In clinical trials, PG was effective as an adjunct to conventional surgery in the treatment of intrabony defects. Because the molecular events due to PG that are able to alter osteob-last activity to promote bone formation are poorly understood, we investigated the expression of os-teoblastic related genes in mesenchymal stem cells exposed to PG. METHODS: The expression levels of bone related genes like RUNX2, SP7, SPP1, COL1A1, COL3A1, BGLAP, ALPL, and FOSL1 and mesenchymal stem cells marker (CD105) were analyzed, using real time reverse transcription-polymerase chain reaction. Pearson's chi-square (χ(2)) test was used to detect markers with significant differences in gene expression. RESULTS: PG caused induction of osteoblast transcriptional factor (like RUNX2), bone related genes osteopontin (SPP1), osteocalcin (BGLAP) and alkaline phosphatase (ALPL). All had statistical sig-nificant P values (< 0.05). CONCLUSION: PG has a differentiation effect on mesenchymal stem cells derived from peripheral blood. The obtained results can be relevant to better understanding of the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

Human cleft lip and palate fibroblasts and normal nicotine-treated fibroblasts show altered in vitro expressions of genes related to molecular signaling pathways and extracellular matrix metabolism.

Nonsyndromic cleft lip with or without cleft palate (CLP) is a frequent craniofacial malformation caused by both genetic and environmental factors. Maternal smoking during pregnancy is a known risk factor, due to the teratogenic role of nicotine. To assess and compare the impact of CLP and nicotine, we studied the quantitative expression of genes involved in signaling pathways and extracellular matrix (ECM) metabolism in human normal nicotine-treated (NicN) and CLP fibroblasts compared to normal control (CTRL) cells. Palatal fibroblast cultures from seven CLP children and seven age-matched CTRL subjects were established and subconfluent cells incubated for 24 h without (CTRL and CLP fibroblasts) or with (NicN fibroblasts) 0.6 mM nicotine. Gene expressions were analyzed by real-time quantitative PCR. For the first time, a regulated cholinergic signaling in our human fibroblasts in vitro was demonstrated. Members of TGF-beta, retinoic acid (RA), and GABA-ergic signaling systems were also differently regulated. Among the ECM genes, fibronectin, syndecan, integrin alpha2, and MMP13 genes were concordantly modulated, while integrin beta5, and decorin genes were discordantly modulated. Interestingly, nicotine treatment regulated gene expressions of CD44 and CLPTM1, two candidate genes for CLP. Our findings show a positive association between nicotine treatment and CLP phenotype. Results suggest that nicotine deranges normal palate development, which might contribute to the development of a CLP malformative phenotype, through the impairment of some important signaling systems and ECM composition.

A comparison between genetic portraits of normal osteoblasts and osteosarcoma cell lines.

BACKGROUND: Osteosarcoma (OS) is the most frequent malignant bone tumor occurring in young patients in the first two decades of life. Metastases are the cause of 90% of cancer deaths for patients with OS. OS of the jaw is rare and aggressive malignancy constitutes approximately 5-13% of all cases of skeletal OS. Chemotherapy plus surgery are the first choice for treatment. AIMS: Because OS cell lines (OCLs) should share a common pathway with primary OS and new drugs are screened in in vitro systems, new insight about the genetic profiling of OCLs is of paramount importance to a better understanding of the molecular mechanism of this rare tumor and detecting a potential target for specific therapy. MATERIALS AND METHODS: The SAOS2 and TE85 cell lines were analysed using DNA microarrays containing 19,000 genes. Several genes in which expression was significantly differentially expressed in OCLs vs. normal osteoblast (NO) were detected. RESULTS: The differentially expressed genes cover a broad range of functional activities: (a) cell cycle regulation, (b) cell differentiation, (c) apoptosis, and (d) immunity. CONCLUSION: The reported data can be relevant to a better understanding of the biology of OS and as a model for comparing the effect of drugs used in OS treatment.

Diphenylhydantoin plays a role in gene expression related to cytoskeleton and protein adhesion in human normal palate fibroblasts.

AIMS: Morphogenetic processes during palate development are related to extracellular matrix composition. The cell-extracellular matrix relation plays a role in cell activity and in gene expression. We studied the effect of diphenylhydantoin, a teratogen known to induce cleft palate in human newborns, on extracellular matrix production. We investigated whether diphenylhydantoin treatment caused any differences in glycosaminoglycans, collagen synthesis and gene expression in human normal palate fibroblasts. METHODS: Human palate fibroblasts were maintained for 24 hours in serum-free 199 medium containing 5 microg/mL (3)H-glucosamine or (3)H proline hydrochloride. Collagen and glycosaminoglycan classes were then measured using biochemical methods, gene expression with microarray analysis and cytoskeleton components with immunofluorescent antibodies and computer analysis. RESULTS: In normal fibroblasts diphenylhydantoin reduced collagen and glycosaminoglycan synthesis with a marked effect on sulphated glycosaminoglycans. There were also substantial decreases in tubulin, vimentin and alpha-actin staining and an increase of vinculin compared to controls. Diphenylhydantoin acted on several genes related to the synthesis of cytoskeleton and adhesion membrane proteins. It inhibited caderin, caveolin, RTK and alpha-actin, and increased nectin, cytoplasmatic FRG vinculin, ITGA, ITGB extracellular matrix ligand and EDG2 gene expression. DNA binding gene expression, which plays a role in cell growth and senescence, was activated. CONCLUSIONS: Since cell activity is dependent on the cell morphology and extracellular matrix composition, these findings indicate that in human normal palate fibroblasts diphenylhydantoin can modify cytoskeletal components and extracellular matrix-cell adhesion, with consequent effects on gene expression. These changes might be related to anomalous palate development.

Gene expression, cytoskeletal changes and extracellular matrix synthesis in human osteoblasts treated with cyclosporin A.

Cyclosporin A (CyA) is an immunosuppressive agent used to prevent allograft rejection, but unfortunately it causes adverse effects such as bone diseases, osteoporosis and osteomalacia. These pathologies involve an imbalance between synthesis, degradation and mineralization of extracellular matrix. CyA can modify extracellular matrix components such as glycosaminoglycans (GAG) and collagen fibers. In addition, normal cell activity is dependent on cell morphology and substrate cell attachment. We treated normal human osteoblasts with CyA and analyzed: (i) gene expression by a microarray method; (ii) extracellular GAG and collagen after (3)H-glucosamine and Western blot analysis; and (iii) cytoskeletal changes, using actin and tubulin fluorescent antibodies. CyA increased intra- and extracellular GAG and extracellular GAG classes such as hyaluronic acid, chondroitin sulphate, and dermatan sulphate; there was no noteworthy effect on heparan sulphate and the ratio of non-sulphated to sulphated GAG. In osteoblast cultures the drug reduced cytoskeletal actin, while tubulin did not change. In vivo the osteoblasts showed morphological changes with different extracellular matrix synthesis. Microarray analysis indicated the inhibition of gene pathways related to Wnt signaling molecules, and the cytoskeletal and focal adhesion cascade. In in vitro human osteoblasts CyA modified gene expression related to cytoskeletal pattern organization and cell morphology. Since in bone pathologies osteoblasts show different morphology related to cell size, these data suggest that in vivo osteoblast different functions could be dependent on alteration of osteoblast differentiation.

Patterns of some extracellular matrix gene expression are similar in cells from cleft lip-palate patients and in human palatal fibroblasts exposed to diazepam in culture.

Prenatal exposure to diazepam, a prototype sedative drug that belongs to Benzodiazepines, can lead to orofacial clefting in human newborns. By using real-time PCR, in the present study we investigated whether diazepam elicits gene expression alterations in extracellular matrix (ECM) components, growth factors and gamma-aminobutyric acid receptor (GABRB3), implicated in the coordinate regulation of palate development. Palate fibroblasts were treated with diazepam (Dz-N fibroblasts) and compared to cleft lip-palate (CLP) fibroblasts obtained from patients with no known exposure to diazepam or other teratogens. Untreated fibroblasts from non-CLP patients were used as control. The results showed significant convergences in gene expression pattern of collagens, fibromodulin, vitronectin, tenascin C, integrins and metalloprotease MMP13 between Dz-N and CLP fibroblasts. Among the growth factors, constitutive Fibroblast Growth Factor 2 (FGF2) was greatly enhanced in Dz-N and CLP fibroblasts and associated with a higher reduction of FGF receptor. Transforming Growth Factor beta 3 (TGFbeta(3)) resulted up-regulated in CLP fibroblasts and decreased in Dz-N fibroblasts. We found phenotypic differences exhibited by Dz-N and CLP fibroblasts in GABRB3 gene regulation, so further studies are necessary to determine whether GABAergic system could be involved in the development of diazepam mediated CLP phenotype. Taken together the results elucidate the molecular mechanisms underlying possible toxicology effects induced by diazepam. Counselling of women on the safety of diazepam exposure is clinically important, also for the forensic consequences.

Genes causing clefting syndromes as candidates for non-syndromic cleft lip with or without cleft palate: a family-based association study.

Clefts of the orofacial region are among the most common congenital defects, caused by abnormal facial development during gestation. Non-syndromic cleft lip with or without cleft palate (NSCLP) is a complex trait most probably caused by multiple interacting loci, with possible additional environmental factors. As facial clefts form part of more than 300 syndromes, one strategy for identifying the genetic causes of NSCLP could be to study candidate genes responsible for clefting syndromes. Three genes were selected for this investigation: TP63, which codes for the tumour protein p63 and causes Ectrodactyly-Ectodermal dysplasia-orofacial Cleft syndrome; JAG2, a downstream gene of TP63; and MID1, which is responsible for Opitz syndrome. A linkage disequilibrium investigation was performed with intragenic single nucleotide polymorphisms on each of these genes in a sample study of 239 patients/parents trios. Evidence which suggests that JAG2 and MID1 may play a role in NSCLP was obtained.

Titanium acts on osteoblast translational process.

Titanium is a highly biocompatible material and very osteogenic in vivo. However, how titanium regulates osteoblast activity to promote bone formation is incompletely characterized. We, therefore, attempted to get more information by using microRNA (miRNA) microarray techniques to investigate translation regulation in osteoblasts grown on titanium disks. The miRNA oligonucleotide microarray provides a novel method to carry out genome-wide miRNA profiling in human samples. By using miRNA microarrays containing 329 probes designed from the human miRNA sequence, several miRNA were identified in osteoblast-like cell line (MG 63) grown on titanium disks. There were 13 upregulated miRNAs (ie, mir-23a, mir-222, mir-523, mir-22, mir-23b, mir-143, mir-377, mir-24, mir-422b, mir-26a, mir-29a, mir-17-5p, mir-182) and 2 down-regulated miRNAs (ie, mir-187, mir-339). The data reported are, to our knowledge, the first study on translation regulation in osteoblasts exposed to titanium. The data can be relevant to understand better the molecular mechanism of osteoblast activation and as a model for comparing other materials with similar clinical effects.

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines.

BACKGROUND: EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF. METHODS: Cell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A. RESULTS: Caco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment. In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases. CONCLUSION: This is the first study to have systematically investigated the effect of cetuximab or gefitinib, alone and in combination with EGF, on human colon cancer cell lines. The EGFR inhibitors have a weaker effect in the presence of EGF that binds EGFR. Cetuximab treatment showed an expression pattern that inversely correlates with EGF treatment. We found interesting cyto-morphological features closely relating to gene expression profile. Both drugs have an effect on differentiation towards cellular death.