FHIT oncosuppressor gene expression profile in human anal cancers.

The FHIT gene, a member of the histidine triad gene family, is a tumor suppressor gene exhibiting deletions in the majority of human cancers. Aberrant transcripts of this gene have been found in about 50% of esophageal, stomach and colon carcinomas. Little is known about the molecular mechanisms involved in malignant transformation of the lining cells of the anus. In this study FHIT gene expression was investigated in this particular kind of human cancer. FHIT expression was comparatively analyzed at the mRNA level, by RT-PCR, in squamous anal cancers, normal anal tissue and peripheral blood samples. cDNA analyses showed variability in FHIT transcripts, without apparent effects on the predicted amino acid sequence. These different FHIT mRNAs could represent transcripts from an alternative splicing event. Our data indicate that the FHIT mRNA detected in anal cancers and in normal samples is heterogeneous. Immunohistochemical data suggest that the Fhit protein is expressed only in a fraction of the tumor cells, while it is strongly expressed in the epithelial cells of glands of the normal anal mucosa. The absence or poor expression of the Fhit protein in anal cancers suggests a role for this tumor suppressor gene product, as a risk factor, in the onset of this human cancer, as reported before for other human gastrointestinal tumors.

Lung regions differently modulate bronchial branching development and extracellular matrix plays a role in regulating the development of chick embryo whole lung.

Normal branching development is dependent on the correlation between cells and extracellular matrix. In this interaction glycosaminoglycans, cytokines and growth factors play a fundamental role. In order to verify the distribution and influence of extracellular matrix and related enzymes on chick embryo lung development, 6 day-old whole lungs were maintained in vitro with testicular hyaluronidase, beta-N-acetyl-D-glucosaminidase and chondrotinase ABC or in linkage with apical, medial and caudal lung regions of 6-day development before and after enzyme treatment. In a separate lung region beta-N-acetyl-D-glucosaminidase and hyaluronidase were determined. Our data show that the whole lung cultures increase bronchial branching development when the medial region is admixed separately, while the separate apical or caudal regions or apical combined with caudal region do not affect bronchial branching development. The enzyme treatment of medial region prevents the branching development in associated whole lung. The bronchial branching development of whole lung cultured in medium containing the enzymes related to glycosaminoglycans turnover is significantly altered. In conclusion, these data show that the different influence of separate apical, medial, caudal lung regions on bronchial branching development is related to the extracellular matrix composition.

Extracellular matrix and growth factors in the pathogenesis of some craniofacial malformations.

The normal development of cranial primordia and orofacial structures involves fundamental processes in which growth, morphogenesis, and cell differentiation take place and interactions between extracellular matrix (ECM) components, growth factors and embryonic tissues are involved. Biochemical and molecular aspects of craniofacial development, such as the biological regulation of normal or premature cranial suture fusion, has just begun to be understood, thanks mainly to studies performed in the last decade. Several mutations has been identified in both syndromic and non-syndromic craniosynostosis patients throwing new light onto the etiology, classification and developmental pathology of these diseases. In the more common craniosynostosis syndromes and other skeletal growth disorders, the mutations were identified in the genes encoding fibroblast growth factor receptor types 1-3 (FGFR1, 2 and 3) where they are dominantly acting and affect specific and important protein binding domain. The unregulated FGF signaling during intramembranous ossification is associated to the Apert and Crouzon syndrome. The non syndromic cleft of the lip and/or palate (CLP) has a more complex genetic background if compared to craniosynostosis syndrome because of the number of involved genes and type of inheritance. Moreover, the influence of environmental factor makes difficult to clarify the primary causes of this malformation. ECM represents cell environment and results mainly composed by collagens, fibronectin, proteoglycans (PG) and hyaluronate (HA). Cooperative effects of ECM and growth factors regulate regional matrix production during the morphogenetic events, connective tissue remodelling and pathological states. In the present review we summarize the studies we performed in the last years to better clarify the role of ECM and growth factors in the etiology and pathogenesis of craniosynostosis and CLP diseases.

SV40 early region and large T antigen in human brain tumors, peripheral blood cells, and sperm fluids from healthy individuals.

SV40 T antigen (Tag) coding sequences were detected by PCR amplification followed by Southern blot hybridization in human brain tumors and tumor cell lines, as well as in peripheral blood cells and sperm fluids of healthy donors. SV40 early region sequences were found in 83% of choroid plexus papillomas, 73% of ependymomas, 47% of astrocytomas, 33% of glioblastoma multiforme cases, 14% of meningiomas, 50% of glioblastoma cell lines, and 33% of astrocytoma cell lines and in 23% of peripheral blood cell samples and 45% of sperm fluids from normal individuals. None of the 13 normal brain tissues were positive for SV40 DNA, nor were seven oligodendrogliomas, two spongioblastomas, one neuroblastoma, one meningioma, or four neuroblastoma cell lines. Expression of SV40 early region was found by reverse transcription PCR, and SV40-specific Tag was detected by indirect immunofluorescence in glioblastoma cell lines. DNA sequence analysis, performed in four positive samples, confirmed that the amplified PCR products belong to the SV40 early region. Sixty-one % of the neoplastic patients positive for SV40 sequences had an age excluding exposure to SV40-contaminated polio vaccines, suggesting a contagious transmission of SV40. The possible role of SV40 Tag in the etiopathogenesis of human brain tumors and the spread of SV40 by horizontal infection in the human population are discussed.

Regulation of protein synthesis by estradiol-17beta in cultured hepatocytes.

Estradiol-17beta added to cultured chick embryo hepatocytes induced the appearance in the medium of a phosphoprotein, identified as phosvitin on the basis of: (i) its behaviour on ionic exchange columns; (ii) its SDS-acrylamide gel electrophoretic mobility; (iii) its amino acid composition. The hormone treatment was also followed by a decreased synthesis of other proteins secreted by the hepatocytes.

Role of the extracellular matrix and growth factors in skull morphogenesis and in the pathogenesis of craniosynostosis.

The complex and largely obscure regulatory processes that underlie ossification and fusion of the sutures during skull morphogenesis are dependent on the conditions of the extracellular microenvironment. The concept that growth factors are involved in the pathophysiology of craniosynostosis due to premature fusion of skull bone sutures, is supported by recent genetic data. Crouzon and Apert syndromes, for example, are characterized by point mutations in the extracellular or transmembrane domains of fibroblast growth factor-2 receptor. In primary cultures of periosteal fibroblasts and osteoblasts obtained from Apert and Crouzon patients, we observed that Crouzon and Apert cells behaved differently with respect to normal cells as regards the expression of cytokines and extracellular matrix (ECM) macromolecule accumulation. Further modulation of ECM components observed after the addition of cytokines provides support for an autocrine involvement of these cytokines in Crouzon and Apert phenotype. Changes in ECM composition could explain the altered osteogenic process and account for pathological variations in cranial development. We suggest that a correlation exists between in vitro phenotype, clinical features and genotype in the two craniosynostotic syndromes. New research into signal transduction pathways should establish further connections between the mutated genotype and the molecular biology of the cellular phenotype.

Interleukin-1 and interleukin-6 differentially regulate the accumulation of newly synthesized extracellular matrix components and the cytokine release by developing chick embryo skin fibroblasts.

In the present study, we demonstrate that both interleukin-1 (IL-1) and interleukin-6 (IL-6) induced a significant decrease in glycosaminoglycan (GAG) synthesis and, more strikingly, secretion by 7 and 13 day-old chick embryo skin fibroblasts. We demonstrated that interleukin treatment also inhibited the synthesis of collagenase-digestible proteins (type I collagen). In addition, tissue culture supernatants (conditioned media, CM) were tested for reactivity for IL specific ELISAs and for their ability to stimulate proliferative responses in mouse thymocytes and hybridoma cells. Our findings demonstrate that chick embryo skin fibroblasts spontaneously produce IL-1 and, in even greater amounts, IL-6. Highest levels of interleukin secretion were found in the CM of 13 day-old fibroblasts and the IL-1 beta isoform was predominant over IL-1 alpha. Pretreatment of the fibroblasts with either IL-1 or IL-6 increased the secretion of both cytokines. Increased IL-1 levels were correlated with enhanced IL-1 bioactivity in the CM of IL-6 treated fibroblasts. By contrast, the raised concentrations of IL-1 in the CM of IL-1 treated cells and IL-6 in the CM of IL-1 or IL-6 treated fibroblasts failed to translate into augmented bioactivity. These observations, taken together, indicated that IL-1 and IL-6 are able to regulate the synthesis and secretion of ECM macromolecules of developing connective tissues and the cytokine release by chick embryo skin fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Maternal MTHFR variant forms increase the risk in offspring of isolated nonsyndromic cleft lip with or without cleft palate.

The pathogenesis of cleft lip with or without cleft palate (CL/P) is complex; its onset could be due to the interaction of various genetic and environmental factors. Recently MTHFR functional polymorphisms were found to increase the risk of this common malformation; however, this finding is still debated. We investigated 110 sporadic CL/P patients, their parents and 289 unrelated controls for c.665C>T (commonly known as 677C>T; p.Ala222Val) and c.1286A>C (known as 1298A>C; p.Glu429Ala) polymorphism in the MTHFR gene. Transmission disequilibrium test (TDT) showed no distortion in allele transmission. Nevertheless, association studies revealed significant differences in allele frequencies between mothers of CL/P patients and controls. This work supports the hypothesis that a lower MTHFR enzyme activity in pregnant women, mostly related to the c.665C>T variant form, is responsible for a higher risk of having CL/P affected offspring.

Interleukin pattern of Apert fibroblasts in vitro.

The phenotype of cultured fibroblasts from patients affected by Apert's syndrome, a rare connective disorder, differs from that of normal cells in its extracellular matrix macromolecule composition (glycosaminoglycans, collagens and fibronectin) and is further modulated by treatment with interleukins (ILs). As the mechanisms responsible for the changes are unknown, we used our recently described model system for Apert periosteal fibroblasts to ascertain whether the pattern of ILs they secrete into the medium is comparable to that of normal fibroblasts. The results obtained by enzyme-linked immunosorbent assay (ELISA) show that the levels of interleukin-1 (IL-1) and interleukin-6 (IL-6) were lower in Apert than in normal media, whereas levels of IL-1 receptor antagonist (IL-1ra), the natural inhibitor of IL-1, were markedly higher. IL-1 specific bio-activity on thymocyte proliferation was also decreased in Apert supernatants. As we provided also evidence that active transforming growth factor beta (TGFbeta1), an IL-1 antagonist, was not secreted in greater amount in Apert media with respect to normals, the enhancement of IL-1ra appeared critical in down-regulating IL-1. Northern blot analysis of cytokine mRNA revealed no detectable IL-1 or IL-6 gene expression in normal fibroblasts, but high amounts of IL-6 mRNA transcripts in Apert cells. As the increased IL-6 gene expression did not translate into a parallel increase of secreted IL-6, the control of IL-6 secretion may be mainly post-transcriptional. Furthermore, the result that a treatment of the cultures with IL-1ra was able to induce a decrease of IL-6 secretion, suggests that the observed decreased secretion of IL-6 may be due to the autocrine action of overproduction of IL-1ra. The observed imbalance in the production of ILs which we show for the first time suggests ILs may be the natural autocrine regulators of ECM production in Apert fibroblasts. We hypothesize that in vitro differences previously reported in fibroblast phenotypes and several clinical features of Apert's syndrome may correlate with different cytokine patterns.

A regulatory role of fibroblast growth factor in the expression of decorin, biglycan, betaglycan and syndecan in osteoblasts from patients with Crouzon's syndrome.

Bone development is controlled by the autocrine and/or paracrine effects of regulatory molecules. We previously showed that the phenotype of fibroblasts obtained from patients affected by Crouzon's syndrome, an autosomal dominant disease characterized by pathological skull bone development, differed from that of normal cells and was regulated by interleukin treatments. The changes in the relative concentrations of extracellular macromolecules (glycosaminoglycans-GAG, collagen and fibronectin) were associated with abnormal interleukin secretion that affected the microenvironment where the osteogenic processes take place. Mutations in human fibroblast growth factor receptors are now thought to be involved in Crouzon's syndrome. Since coactivation of interleukins and basic fibroblast growth factor (bFGF) is probably implicated in morphogenetic and osteogenic processes and heparan sulphate proteoglycans have a critical role in regulating bFGF activity, the phenotypes of normal and Crouzon osteoblasts were studied and the effects of bFGF on the expression of bFGF, procollagen alpha1 (I), and proteoglycan (PG) genes for biglycan, decorin, betaglycan and syndecan analyzed. Specific human cDNA probes were used to screen the relative levels of mRNA by Northern analysis. Spontaneous or bFGF-modulated release of interleukins was also assayed. The bFGF gene transcript was detected only in Crouzon osteoblasts. We showed for the first time that Crouzon osteoblasts, despite a mutation in the FGF receptor, still responded to exogenous bFGE In fact, the growth factor induced changes in the GAG profile and in the levels of mRNA coding for PG and procollagen alpha1 (I) and down-regulated heparan sulfate GAG chains. ELISA showed that bFGF-induced interleukin secretion differed in normal and Crouzon osteoblasts. The observed differences in PG core protein, procollagen alpha1 (I) and bFGF could be associated with the Crouzon bone phenotype and also should provide further understanding on the molecular basis of the diseased state of bone.

The murine DSCR1-like (Down syndrome candidate region 1) gene family: conserved synteny with the human orthologous genes.

A recently recognized gene family, conserved from yeast to humans, includes Down syndrome candidate region 1 gene (DSCR1), Adapt78 (recognized as the hamster ortholog of the DSCR1 isoform 4), ZAKI-4 (renamed DSCR1-like 1, DSCR1L1) and DSCR1L2 (a novel gene on human chromosome 1), along with yeast and C. elegans single members (Strippoli P., Lenzi L., Petrini M., Carinci P., Zannotti M., 2000. A new gene family including DSCR1 (Down Syndrome Candidate Region 1) and ZAKI-4: characterization from yeast to human and identification of DSCR1-like 2, a novel human member. Genomics 64, 252-263). The proposed family labels were a putative single-strand nucleic acid binding domain similar to the RNA recognition motif, and a unique, highly-conserved serine-proline motif. We have used a bioinformatics-driven molecular biology approach to characterize the murine members of DSCR1-like gene family. Systematic expressed-sequence-tags (EST) database search and reverse-transcription polymerase chain rection (RT-PCR) product sequencing allowed identification of the murine DSCR1, DSCR1L1 and DSCR1L2. The sequences of the respective protein products are of 198, 197 and 241 amino acids, respectively, and are very similar to the corresponding human proteins. The very broad expression pattern of the murine DSCR1 genes is similar to that of the human genes. Using a radiation hybrid panel, we mapped the murine DSCR1-like family members. The murine DSCR1 ortholog is located on the chromosome 16, in a region corresponding to that on human chromosome 21 just upstream of the Down syndrome candidate region. DSCR1L1 and DSCR1L2 murine genes are also located in chromosomal segments of chromosome 17 and 4, respectively, exactly corresponding to those containing the respective human homologs on chromosomes 6 and 1. Description of the mouse orthologs for DSCR1-like genes will allow knockout mice to be obtained for specific family members.

Bronchial branching correlates with specific glycosidase activity, extracellular glycosaminoglycan accumulation, TGF beta(2), and IL-1 localization during chick embryo lung development.

During organ differentiation, cell-extracellular matrix (ECM) interactions are required. The components of the ECM, such as glycosaminoglycans, fibronectin, laminin, and collagens, change in relation to cytokine and enzyme activity. Moreover, glycosaminoglycans (GAGs) are components of the ECM that play an important role in both cytokine regulation and cell activities. In this work we studied the accumulation of hyaluronic acid and chondroitin sulfate and heparan sulfate proteoglycans (PGs), beta-N-acetyl-D-glucosaminidase activity, the presence of transforming growth factor beta(2) (TGF beta(2)), and interleukin-1 (IL-1), and the localization of fibronectin, laminin, and collagen I and IV during the early stages of chick embryo lung development. We also determined the levels of hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate GAGs and the activity of beta-N-acetyl-D-glucosaminidase with biochemical methods. Our data show that beta-N-acetyl-D-glucosaminidase activity increases in each cell, especially in the epithelial growth front at the emergence of each bronchial bud, where hyaluronic acid and IL-1 are located in the surrounding mesenchymal areas. Chondroitin sulfate and heparan sulfate PGs, fibronectin, laminin, and collagen I and IV are evident in the area near the basal membrane along the sides where the forming structures are stabilized. Biochemical data show that beta-N-acetyl-D-glucosaminidase activity increases in cells during lung development and is related to GAG decrease and to modifications of the nonsulfated/sulfated GAG ratio. These modifications could change cytokine activity and play an important role in bronchial branching development.

Lack of linkage disequilibrium between transforming growth factor alpha Taq I polymorphism and cleft lip with or without cleft palate in families from Northeastern Italy.

Cleft lip with or without cleft palate (CL +/- P) is the most frequent craniofacial malformation in different human populations and its cause is largely unknown. Several studies based on population associations have suggested that an allele mapping in the transforming growth factor alpha locus could be responsible, as a risk factor, for the development of the defect. Our investigation of the Taq I polymorphism at the transforming growth factor alpha locus, performed in 40 CL +/- P families, did not find evidence for linkage disequilibrium with particular alleles. Moreover, tight linkage was excluded with the traditional LOD score method.

C677T variant form at the MTHFR gene and CL/P: a risk factor for mothers?

Maternal folic acid supplementation in early pregnancy has been suggested to play a role in the prevention of nonsyndromic orofacial cleft, i.e., cleft lip with or without cleft palate (CL/P). Moreover, some authors demonstrated association of the C-->T mutation (C677T), converting an alanine to a valine residue in 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, with other congenital anomalies such as neural tube defects (NTDs). Because of MTHFR's involvement in the metabolism of folate, we investigated 64 CL/P patients and their parents for C677T MTHFR mutation. No linkage disequilibrium was found using the transmission disequilibrium test (TDT). However, a significantly higher mutation frequency was detected in mothers of CL/P patients compared to controls. The odds ratios calculated for mothers having CT or TT genotype, compared to the normal CC genotype, were 2.75 (95% confidence interval 1.30-5.57) and 2.51 (1.00-6.14), respectively. These results support the involvement of the folate pathway in the etiology of CL/P, and indicate an effect of the maternal genotype, rather than influence of the embryo's genotype.

Expression analysis and mutational screening of the epithelium-specific ets gene-1 (ESE-1) in patients with squamous anal cancer.

To investigate whether ESE-1 gene abnormalities are involved in alterations of epithelial cell differentiation in squamous anal cancer ESE-1 expression and structure were screened in six patients by reverse transcriptase-polymerase chain reaction (RT-PCR) and automated sequence analysis. The complete cDNA of isoform ESE-1b was always expressed and correctly spliced, with single nucleotide polymorphism being observed in two cases. Presence of ESE-1b point mutations was excluded. Expression of SPRR2A and ENDOA/CK8, two epithelium-specific ESE-1 target genes, were revealed by RT-PCR in all cases. This first report of expression of ESE-1, and of SPRR2A and ENDOA/CK8 (both related to terminal differentiation in different types of epithelia lining) in anal cancer excludes the hypothesis that these genes influenced carcinogenesis in our patients. Despite selecting of patients without clinical evidence of HPV infection, PCR consistently revealed HPV-16 DNA, highlighting the importance of HPV infection in anal cancer.

Expression profile of epidermal differentiation complex genes in normal and anal cancer cells.

Anal cancer originates from a peculiar histological region and provides a useful model for investigating alterations in proliferation and/or differentiation of neoplastic keratinocytes. Epidermal differentiation complex (EDC) genes, which form one of the major gene clusters in the human genome, are involved in the terminal differentiation of epithelial cells and in many instances have been implicated in epithelial tumours. We constructed a DNA macroarray capable of characterising the expression profiles of the entire EDC gene complex in normal mucosa and anal cancer biopsies of seven unrelated patients. Brain tissue and cultured keratinocytes were used as controls. All anal cancer samples showed expression profiles in which none of the EDC genes was silent, as evaluated by phosphor-imager analysis. Variance analysis showed significantly lower expression of SPRR2 with respect to SPRR1 or SPRR3, and significantly higher expression of S100A8 than of other S100A subfamily members. At hierarchical clustering analysis, the four basaloid anal cancer cases conglomerated in the top five positions. The macroarray method used by us provides the first demonstration of the expression profile of the EDC gene family in anal cancer, and is capable of producing significant information on the subgrouping of epithelial tumours such as anal cancer.

Interactions of fibroblasts with soldered and laser-welded joints.

Relatively little is known about the biocompatibility of the soldered or laser-welded joints of dental appliances. We investigated the reaction of human gingival fibroblasts cultured in vitro in direct contact with samples of soldered and laser-welded joints from orthodontic lingual arches. Contrast phase light microscopy was used to evaluate cell adhesion, morphology and proliferation after 6 and 24h and after 7 and 16 days. Scanning electron microscopy (SEM) was performed at 16 days. Our in vitro findings provide evidence that laser-welded orthodontic appliances have superior fibroblast biocompatibility.

Diphenylhydantoin affects glycosaminoglycans and collagen production by human fibroblasts from cleft palate patients.

During embryonic development, the proper production of extracellular matrix molecules mediates morphogenetic processes involved in palatogenesis. In the present study, we investigated whether any differences exist in glycosaminoglycan (GAG) and collagen synthesis between palate fibroblasts from infants, with or without cleft palate, in two age ranges. Subsequently, the effects of diphenylhydantoin (PHT), a teratogen known to induce cleft palate in human and mammalian newborns, on extracellular matrix (ECM) production were studied. We found that cleft palate fibroblasts (CPFs) synthesize greater amounts of GAG and collagen than normal fibroblasts (NFs). CPFs produced less cellular hyaluronic acid (HA) and more sulphated GAG. HA was the principal GAG species in the medium, and its percentage was lower in one- to three-year-old CPFs. Cleft palate fibroblasts produced more extracellular chondroitin 4- and 6-sulphate (CS) and dermatan sulphate (DS). Associated with a higher production of sulphated GAG, we observed a higher synthesis of type III and type I collagen with a normal ratio of alpha2(I) to alpha1(I) chains. PHT treatment of NFs reduced collagen and GAG synthesis, with a marked effect on sulphated GAG. The drug changed collagen synthesis, whereas it did not affect GAG production in CPFs whose phenotype may already be impaired. These findings indicate that, in CPFs, modifications in the pattern of ECM components, which are most likely responsible for the anomalous development, persist in infants. In addition, NFs and CPFs with a different phenotype respond differently to PHT treatment.

Genetic expression profiling of six odontogenic tumors.

Odontogenic tumors are rare neoplasms arising from the odontogenic apparatus. We aimed to identify molecular characteristics associated with odontogenic tumorigenesis and malignancy. To this end, we investigated the expression level of human genes by using, for the first time in odontogenic tumors, the technique of expression profiling. Gene expression alterations common to all six odontogenic tumors were identified by the use of cDNA microarrays containing 19,000 human cDNAs. Statistical analysis on a subset of 4974 cDNAs present in the biopsies identified 506 distinct genes associated with the tumors (p-value < 0.01). Gene ontology analysis of the cellular processes which were differentially regulated in odontogenic tumors was accomplished by the use of a subset of 1409 annotated genes. Finally, 43 cDNAs differentiated the three malignant odontogenic tumors (ameloblastic carcinoma, clear cell odontogenic tumor, granular cell odontogenic tumor) from the three benign ameloblastoma biopsies (p < 0.01). The identified genes might help us better classify borderline odontogenic tumors.

TGFbeta isoforms and decorin gene expression are modified in fibroblasts obtained from non-syndromic cleft lip and palate subjects.

Interaction between extracellular matrix (ECM) and cytokines is thought to be crucial for palatal development. The localization of transforming growth factors (TGFalpha and TGFbeta isoforms) in craniofacial tissues suggests that they carry out multiple functions during development. In the present report, we studied TGFalpha, TGFbeta1, and TGFbeta3 expressions and their effects on ECM macromolecule production of normal and cleft palatal fibroblasts in vitro, to investigate the mechanisms by which the phenotypic modulation of fibroblasts occurs during the cleft palate process. The results indicated that, while TGFalpha mRNA was not evidenced in CLP or normal fibroblasts, a reduced TGFbeta1 hybridization signal was detected in CLP fibroblasts. In addition, these secreted more active TGFbeta3 than TGFbeta1, as evaluated in a biological assay. The CLP phenotype, which differed from the normal one because of its higher PG decorin expression and greater production of GAG and collagen, was further modified by the addition of growth factors. In fact, in CLP fibroblasts, TGFalpha and TGFbeta1 down-regulated PG decorin transcript, TGFbeta1 increased collagen and GAG in both cellular and extracellular compartments, and TGFbeta3 promoted secretory processes of cells. In conclusion, the data represent the first report in a human model in vitro that TGFbeta1 and beta3 are differently expressed and are correlated to the CLP phenotype. Thus, strength is given to the hypothesis that TGFbeta isoforms are the potential inducers of phenotypic expression in palatal fibroblasts during development and that an autocrine growth factor production mechanism may be responsible for the phenotypic modifications.