We report on a male twin infant who presented with brachy-turri-cephaly, frontal bossing, large anterior fontanelle, low set and malformed ears, and mild arachnodactyly. He had normal male genitalia. There was no evidence for maternal virilization during pregnancy. The pattern of malformations resembled Antley-Bixler-Syndrome (ABS). However, sequencing analysis of the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 gene (FGFR2) did not reveal mutations. The boy's twin sister did not show any somatic or endocrine abnormalities. In the boy, neonatal screening for congenital adrenal hyperplasia was positive with moderately elevated <em>17</em>-hydroxyprogesterone. Sequence analysis of his CYP21 gene did not reveal any mutations. The short synacthen test revealed an exaggerated <em>17</em>-hydroxyprogesterone and a blunted cortisol response. Urinary steroid profiling by gas chromatography-mass spectrometry (GC-MS) revealed a unique steroid metabolome suggestive of impaired activity of both <em>17</em>-hydroxylase and 21-hydroxylase. Clinical and metabolic findings therefore were compatible with the recently described variant of congenital adrenal hyperplasia, P450 oxidoreductase deficiency (ORD). Subsequently, sequencing analysis of CPR, the gene encoding P450 oxidoreductase (OR), revealed a homozygous mutation in the patient, resulting in an amino acid exchange in position 284 of the OR protein (A284P). Both the female twin sister and the parents were heterozygous for the A284P mutation. P450 oxidoreductase deficiency represents a novel autosomal recessively inherited form of congenital adrenal hyperplasia. Its characteristic steroid metabolome can readily be detected by GC-MS analysis of spot urine. Clinical features may include an ABS phenotype, ambiguous genitalia (virilization in girls, feminization in boys), and glucocorticoid deficiency. If required, hydrocortisone replacement should be provided.

The vaccinia-related kinases (VRKs) are highly conserved throughout the animal kingdom and phosphorylate several chromatin proteins and transcription <em>factors</em>. In early Caenorhabditis elegans embryos, VRK-1 is required for proper nuclear envelope formation. In this work, we present the first investigation of the developmental role of VRKs by means of a novel C. elegans vrk-1 mutant allele. We found that VRK-1 is essential in hermaphrodites for formation of the vulva, uterus, and utse and for development and maintenance of the somatic gonad and thus the germ line. VRK-1 regulates anchor cell polarity and the timing of anchor cell invasion through the basement membranes separating vulval and somatic gonadal cells during the L3 larval stage. VRK-1 is also required for proper specification and proliferation of uterine cells and sex myoblasts. Expression of the <em>fibroblast</em> <em>growth</em> <em>factor</em>-like protein EGL-<em>17</em> and its receptor EGL-15 is reduced in vrk-1 mutants, suggesting that VRK-1 might act at least partially through activation of FGF signaling. Expression of a translational VRK-1Colon, two colonsGFP fusion protein in the ventral nerve cord and vulva precursor cells restores vulva and uterus formation, suggesting both cell autonomous and non-autonomous roles of VRK-1.

The insulin-like <em>growth</em> <em>factors</em> (IGF-I and IGF-II), their receptors and binding proteins (IGFBPs) are endogenously expressed in a number of tissues including the lung during fetal and neonatal development. This endogenous autocrine/paracrine IGF 'system', together with endocrine sources, contributes to the regulation of lung cell proliferation. We investigated the expression of the mRNAs encoding IGF-I, IGF-II, the type 1 IGF receptor (IGF-T1R) and two IGF-binding proteins (IGFBP-2 and IGFBP-4) in rat lung during the perinatum. These were compared in lung with surfactant apoprotein A (Sp-A) mRNA levels. mRNA in extracts of fetal tissues collected between day <em>17</em> of gestation (<em>17</em>f) and day 9 after birth (9d) was estimated by Northern blot or RNase protection analysis. At day 20 of gestation IGF-I, IGF-T1R and IGFBP-4 mRNA levels were higher in lung than liver (all P < 0.01), whereas IGF-II and IGFBP-2 mRNA levels were higher in liver than lung (each P < 0.02). The expression of IGF-1, IGFBP-2 and IGFBP-4 in lung was high before birth (days <em>17</em>-20f) but decreased to low levels at days 21f, 22f or at birth (1d) but increased in the neonatal lung. IGF-II expression in lung was high at <em>17</em>f but decreased before birth and remained low after birth. The IGF-T1R was expressed at moderate levels before birth, decrease before birth but peaked at days 2-5 after birth. The decrease in expression of these <em>growth</em> regulators before birth expression of these <em>growth</em> regulators before birth was matched by an increased in Sp-A expression which was clearly seen at day 20f, peaked at 1d and then was clearly seen at day 20f, peaked at 1d and then was maintained at high levels after birth. Primary cell cultures of 18f lung epithelia express IGFBP-2 while <em>fibroblasts</em> from the same animals express only IGFBP-4. Cells grown from 22f lung tissue express IGFBP-2 and IGFBP-4 at lower levels, behaving in vitro as they do in vivo. The contrasting levels of expression of different components of the IGF system in the fetal lung and liver indicate organ-specific regulation. IGFBP-2 and IGFBP-4 expression in different cell types within lung but with similar temporal changes suggests cell-specific regulation, perhaps by a common agent. The patterns by a common agent. The patterns of expression of IGF-I, IGF-T1R, IGFBP-2 and IGFBP-4, but not IGF-II, in developing lung correspond to previously described phasic changes in lung cell proliferation rates. The nadir in expression of these four major components of the lung IGF system occurs in the saccular phase when the lung begins to differentiate, probably under the influence of certain endocrine agents.

The therapeutical interest of pluripotent cells and ethical issues related to the establishment of human embryonic stem cell (ESC) or embryonic germ cell (EGC) lines raise the understanding of the mechanism underlying pluripotency to a fundamental issue. Establishing a protein pluripotency signature for these cells can be complicated by the presence of unrelated proteins produced by the culture environment. Here, we have analyzed the environment supporting ESC and EGC <em>growth</em>, and established 2-D reference maps for each constituent present in this culture environment: mouse embryonic <em>fibroblast</em> feeder cells, culture medium (CM) and gelatin. The establishment of these reference maps is essential prior to the study of ESC and EGC specific proteomes. Indeed, these maps can be subtracted from ESC or EGC maps to allow focusing on spots specific for ESCs or EGCs. Our study led to the identification of 110 unique proteins from <em>fibroblast</em> feeder cells and 23 unique proteins from the CM, which represent major contaminants of ESC and EGC proteomes. For gelatin, no collagen-specific proteins were identified, most likely due to difficulties in resolution and low quantities. Furthermore, no differences were observed between naive and conditioned CM. Finally, we compared these reference maps to ESC 2-D gels and isolated <em>17</em> ESC specific spots. Among these spots, proteins that had already been identified in previous human and mouse ESC proteomes were identified but no apparent ESC-specific pluripotency marker could be identified. This work represents an essential step in furthering the knowledge of environmental <em>factors</em> supporting ESC and EGC <em>growth</em>.

Although several reports have indicated a role for endoderm in the regulation of heart development, the mechanism remains unknown. To begin characterization of endoderm-secreted proteins, explants from postgastrulation (Hamburger-Hamilton stage 5/6-8) chicken embryos were cultured in defined medium. Fluorography of SDS-PAGE gels revealed a pattern of synthesized, secreted proteins that was independent of time in culture or embryonic stage when explants were removed. Approximately 10 labeled bands were detected, the most prominent of which migrated at <em>17</em>, 25, and 200 kDa. ELISA analysis revealed that while acidic and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>-like antigens were barely detectable, fibronectin and inhibin beta A were very reactive. Western blot analysis verified the presence of fibronectin and, most remarkably, inhibin beta A, activin dimers of which have recently been implicated in Xenopus mesoderm induction (Smith, Price, Van Nimmen, and Huylebrock (1990). Nature 345, 729.)

Nerve <em>growth</em> <em>factor</em> (NGF) is found in high concentrations in the mouse salivary gland. However, this gland is unique since salivary glands from other animals have only trace amounts of NGF. In the mouse gland, two high molecular weight forms of NGF have been reported, 7S-NGF [Varon, S., Nomura, J., & Shooter, E.M. (1967) Biochemistry 6, 2202-2209] and NGF1 [Young, M., Saide, J.D., Murphy, R.A., & Blanchard, M.H. (1978) Biochemistry <em>17</em>, 1490-1498]. 7S-NGF is comprised of three noncovalently associated subunits: beta-NGF, which is the biologically active subunit, alpha subunit, and gamma subunit. A similar subunit composition is seen with NGF1 (unpublished work with R.A. Murphy). Since the mouse salivary gland is unique with regard to its synthesis of NGF, the following question arises. Do other sources of NGF produce either 7S-NGF or NGF1? Mouse <em>fibroblast</em> cells (L929) in culture synthesize and secrete into their feeding medium (conditioned medium) a beta-NGF-like molecule [Pantazis, N.J., Blanchard, M.H., Arnason, B.G.W., & Young, M. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1492-1496]. These cells therefore provided the opportunity to examine the molecular nature of NGF produced by a nonsalivary gland source. In this study, it was determined by radioimmunoassay that neither the alpha nor the gamma subunit is present in <em>fibroblast</em> cell conditioned medium. Since alpha- and gamma-proteins are present in both 7S-NGF and NGF1, this indicates that neither of the salivary gland forms of NGF are produced by the mouse <em>fibroblast</em> cell.

We have established recently a series of unique cell lines (NS series) from dispase-separated mouse epidermis that promote the <em>growth</em> of epidermal-derived antigen-presenting cell lines (XS series). The purposes of this study were to determine the identity of NS cells and to characterize their functional properties. NS cells were distinguishable from leukocytes by the lack of typical surface markers and by the failure to respond to leukocyte <em>growth</em> <em>factors</em>. Despite their epidermal derivation, NS cells were distinct from keratinocytes by the absence of cytokeratins. On the other hand, NS cells were indistinguishable from lines of dermal <em>fibroblasts</em> by their a) morphology, b) surface phenotype, and c) intracellular deposits of type I collagen. <em>Growth</em> of the XS antigen-presenting lines has been promoted by co-culturing with gamma-irradiated NS cells, and this activity could be replaced with NS cell-conditioned media alone, but not with paraformaldehyde-fixed NS cells. Each clone derived from the NS01 line secreted XS cell-<em>growth</em>-promoting activity, and this activity was blocked by monoclonal antibodies against colony-stimulating <em>factor</em>-1 receptors. Dermal <em>fibroblasts</em> also promoted the <em>growth</em> of XS cells in a colony-stimulating <em>factor</em>-1-dependent manner. By contrast, culture supernatants from other cell lines derived from skin (e.g., Pam 212 keratinocytes, 7-<em>17</em> dendritic epidermal T cells, or XS lines) failed to promote XS cell <em>growth</em>. These results indicate that NS cells belong to the <em>fibroblast</em> lineage and that they share the intrinsic property to secrete large amounts of colony-stimulating <em>factor</em>-1 with dermal <em>fibroblasts</em>. Dermal cells may support the <em>growth</em> of skin-associated antigen-presenting cells in vivo.

OBJECTIVE

Tissue inhibitors of metalloproteinases (TIMPs) are multi-functional proteins with matrix metalloproteinases-inhibiting activities. We studied expression of anti-inflammatory, TIMP-4 gene in human joint tissues and its regulation by arthritis-associated cytokines.

RESULTS

TIMP-4 RNA expression originating from synovial <em>fibroblasts</em> was significantly (2.4 fold; p<0.001) elevated in 8 osteoarthritic (OA) versus 7 non-arthritic synovial membranes. Non-arthritic and OA femoral head and knee chondrocytes displayed substantial but variably constitutive expression of the TIMP-4 mRNA. In articular chondrocytes, transforming <em>growth</em> <em>factor</em> beta (TGF-β1) and oncostatin M (OSM) upregulated TIMP-4 RNA and protein expression while interleukin-1 (IL-1β) and tumor necrosis <em>factor</em> alpha (TNF-α) did not, suggesting differential regulation by arthritis-associated cytokines. Interleukin <em>17</em> (IL-<em>17</em>) mildly induced TIMP-4 mRNA. TGF-β1 induction of TIMP-4 expression was partly inhibited by ERK pathway and Sp1 transcription <em>factor</em> inhibitors.

CONCLUSIONS

Enhanced TIMP-4 gene expression in OA synovial membranes and cartilage may be due to induction by TGF-β1, OSM and IL-<em>17</em>, suggesting its pathophysiological role in tissue remodeling in human joints. TGF-β1 induction of TIMP-4 expression is mediated partly by ERK pathway and Sp1 transcription <em>factor</em>.

OBJECTIVE

Thymidine phosphorylase (TP) in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) is induced by tumor necrosis factor α (TNFα) and other cytokines that have been reported to be major inflammation mediators in RA. We previously demonstrated that TP plays an important role in angiogenesis and tumor growth, invasion, and metastasis. The aim of this study was to investigate whether the role of TP in the pathogenesis of RA is similar to its role in tumors.

METHODS

In FLS obtained from 2 patients with RA, the expression of TP, interferon-γ (IFNγ)-inducible protein 10 (CXCL10), and other cytokines was measured by quantitative real-time polymerase chain reaction, immunoblotting, and enzyme-linked immunosorbent assays. Microarray analysis was performed using FLS transfected with TYMP complementary DNA and treated with a TP inhibitor.

RESULTS

The expression of TP in FLS was up-regulated by TNFα, interleukin-1β (IL-1β), IL-17, IFNγ, and lipopolysaccharide. Microarray analysis of FLS overexpressing TP identified CXCL10 as a thymidine phosphorylase-related gene. The expression of CXCL10 was induced by TNFα, and this induction was suppressed by TYMP small interfering RNA and TP inhibitor. Furthermore, the combination of TNFα and IFNγ synergistically augmented the expression of TP and CXCL10. TP-induced CXCL10 expression was suppressed by the antioxidant EUK-8. In the synovial tissue of patients with RA, TP levels were significantly correlated with CXCL10 expression.

CONCLUSIONS

The combination of TNFα and IFNγ strongly induced the expression of thymidine phosphorylase in RA FLS. The induction of thymidine phosphorylase enhanced the expression of CXCL10, which may contribute to the Th1 phenotype and bone destruction observed in RA.

Adrenocortical cell major secreted protein was purified from the conditioned medium of primary cultures of bovine adrenocortical (BAC) cells. Immunochemical analysis and N-terminal sequencing of the purified protein identified it to alpha 2-macroglobulin (alpha 2-M). It appeared that 15 out of the <em>17</em> N-terminal amino acids were conserved between adrenocortical cell major secreted protein and human alpha 2-M. Study of alpha 2-M production by BAC cells revealed that its secretion was stimulated severalfold by transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta 1). The stimulation occurred in a time-dependent (reaching a plateau at 24 h) and dose-dependent (ED50 = 0.1 ng/ml TGF-beta 1) manner. It was blocked when BAC cells were exposed to 5,6-dichlorobenzimidazole riboside, a potent inhibitor of RNA polymerase II, suggesting that TGF-beta 1 acts as an activator of alpha 2-M gene expression at the transcriptional level. Northern blot analysis confirmed that the alpha 2-M mRNA level was increased (4-fold) in BAC cells following TGF-beta 1 treatment. TGF-beta 2, TGF-beta 1,2, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and angiotensin II also appeared able to stimulate alpha 2-M secretion in BAC cells, whereas adrenocorticotropin was strongly inhibitory. Given the previous reports that TGF-beta 1 is a potent inhibitor of adrenocortical steroidogenesis (Feige J.J., Cochet, C., Rainey, W.E., Madani, C., and Chambaz, E. M. (1987) J. Biol. Chem. 262, 13491-13495) and that alpha 2-M is a TGF-beta 1-binding protein, these observations suggest that alpha 2-M may play an important role in conjunction with hormones and <em>growth</em> <em>factors</em> in the homeostatic regulation of adrenocortical functions.

BACKGROUND

Angiogenesis plays a significant role in the growth and progress of cancer, thus we evaluated the levels of urinary basic fibroblast growth factor (bFGF) in bladder cancer (Ca) patients and investigated any possible correlation between this angiogenic factor with tumor stage and grade.

METHODS

Urine samples from 41 patients with bladder Ca, 11 patients with history of bladder Ca but negative follow-up cystoscopy, 18 patients with benign prostate hyperplasia (BPH) and 15 normal healthy volunteers were assayed using an enzyme-linked immunosorbent assay for bFGF. Resulting values were normalized against urine creatinine and expressed as pg/g.

RESULTS

The median urinary bFGF level of patients with active disease, history of bladder carcinoma and negative follow-up cystoscopy, BPH, and healthy volunteers were 2,717, 1,009, 1,414 and 1,100 pg/g, respectively. There was a statistically significant difference between median bFGF levels of patients with active bladder Ca and those of the other groups (p = 0.000). Eleven patients with invasive bladder Ca had a median bFGF value of 6,880 pg/g that was significantly increased (p = 0.002) compared to the median of 2,312 pg/g of those with superficial tumors (Ta 14, T1 16). Grades 1, 2 and 3 carcinoma were found in 5, 19 and 17 patients which had a median bFGF of 2,717, 1,762 and 3,617 pg/g, respectively, but the difference was not statistically significant (p = 0.13).

CONCLUSIONS

Our results confirm the implication of bFGF in the biology of bladder cancer, and demonstrate that urinary bFGF concentration seems to be significantly related to the stage but not to the grade of the disease supporting the proposed mechanisms of release of bFGF. Further studies are required in order to validate the potential clinical applications of bFGF for specific groups of bladder cancer patients.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) significantly enhances the short-term survival of embryonic striatal neurons in vitro but has little effect on the out<em>growth</em> of striatal cells compared to neurons from other brain regions. Studies in our laboratory have shown that bFGF protects postnatal striatal cells in vitro from NMDA receptor-induced neurotoxicity. We therefore examined the effects of bFGF on the out<em>growth</em> of GABA-containing cells taken from the postnatal (Day 1) caudate-putamen and cultured for up to 3 weeks. In control cultures GABAergic neurons formed three populations based on somatic size and developed the cytoarchitectural features characteristic of dendrites, spines, and axons. In the presence of bFGF (6 pM continuously from the day of plating), small- and medium-sized GABAergic neurons showed significant increases compared to untreated controls in axon-like <em>growth</em> (axon length) at 6 days in culture and in both axon- and dendrite-like neurite <em>growth</em> (axon length and branch order, number of primary dendrites, dendrite length, and dendritic branch order) at 13 and <em>17</em> days in culture. Large GABAergic neurons were unaffected by treatment with bFGF. Striatal GABAergic neurons exposed to nerve <em>growth</em> <em>factor</em> (10 ng/ml) were not different from untreated controls. Neuron survival was also unaffected by bFGF treatment at all days in culture examined. Other observations suggested that the neurotrophic effects of bFGF were mediated by a direct action of the <em>growth</em> <em>factor</em> on striatal neurons and not glial cells. First, glial cells (identified by the immunohistochemical localization of glial fibrillary acidic protein) were unaffected by bFGF treatment at the low concentration (6 pM) used to enhance neurite <em>growth</em>, but did significantly proliferate at higher concentrations of bFGF (6 nM). Second, immunoreactive bFGF receptor protein was localized predominantly to the somata and processes of striatal neurons and not to glial cells in the cultures. Finally, when neurons from control cultures were briefly exposed (1 to 4 h) to bFGF at concentrations which were neurotrophic, a marked elevation in the immediate early gene protein c-fos was observed by immunohistochemistry in the nuclei of neurons, including GABAergic cells.(ABSTRACT TRUNCATED AT 400 WORDS)

The cytokine transforming <em>growth</em> <em>factor</em>-beta has multiple effects on a wide variety of cell types. These effects include modulation of <em>growth</em> and regulation of gene transcription. In the present work, we demonstrate that TGF-beta1 increases transcription of the latent transforming <em>growth</em> <em>factor</em>-beta binding protein-2 ( LTBP-2) gene in cultured human fetal lung <em>fibroblasts</em> leading to a significant increase in LTBP-2 mRNA steady state level. The stability of LTBP-2 mRNA was not appreciably altered. A corresponding increase in production of LTBP-2 protein accompanied the increase in mRNA. Through the use of specific inhibitors, we demonstrate that a member of the Ras super family and a protein kinase C, probably of the atypical (non-diacylglycerol, non-Ca++ dependent) class are likely to be components in the signaling pathway. However, phospholipases, G proteins and extracellular-signal regulated kinases do not appear to be involved. These results combined with previous findings on elastin regulation by TGF-beta1 (Kucich et al. (1997). Am. J. Respir. Cell Mol. Biol., <em>17</em>: 10-16) demonstrate that TGF-beta1 can coordinately increase the steady state levels of mRNAs encoding components of the elastic fiber, but through diverse mechanisms. In contrast to LTBP-2, increased elastin expression is achieved by message stabilization. Furthermore, the TGF-beta1 signaling pathways differ and while the pathway leading to increased LTBP-2 transcription shares components with those modulating transcription of other genes, it is unlikely to be precisely congruent with any other previously described one.

Estrogens have been shown to exert significant benefits on the cardiovascular system both in animals and in postmenopausal women. However, the exact mechanism of these effects are, for the most part, still unknown. The goal of this paper is to evaluate the role of estrogen receptors (ER) in mediating some of the cardiovascular beneficial actions of <em>17</em> beta-estradiol (E2). This analysis was possible because of the availability of ER alpha (ER alpha KO) and ER beta-deficient (ER beta KO) mice, and access to a patient with ER alpha-deficiency. Experimental results obtained in our laboratory demonstrated that the ER alpha subtype mediates E2-induced increase in endothelial nitric oxide production and facilitation of <em>fibroblast</em> <em>growth</em> <em>factor</em>-elicited angiogenesis in vivo. Others have confirmed these findings. Experiments using a novel ER-antagonist and ApoExER alpha double-knockout mice proved that ER alpha mediates some of the antiatherosclerotic effects of E2 as well. In contrast, both the ER alpha and ER beta subtypes appear to mediate the beneficial effects of E2 on vascular smooth muscle proliferation after vessel injury. The young male patient with ER alpha-deficiency exhibited reduced endothelial nitric oxide production and premature coronary arteriosclerosis. These studies in mice and a male human subject suggest that absence of functional ER may represent a novel risk <em>factor</em> for cardiovascular diseases.

BACKGROUND

Although mechanical vascular injury leads to smooth muscle cell proliferation that contributes to restenosis after balloon angioplasty, the role of the single transient mechanical stimulation of smooth muscle cells in this process is unknown.

RESULTS

To test the hypothesis that a single transient mechanical stimulus can increase DNA synthesis, human vascular smooth muscle cells cultured in a three-dimensional collagen gel system were subjected to transient compression. Transient compression (5-minute duration) of smooth muscle cell-collagen gel cultures in defined serum-free conditions led to delayed increases in [3H]thymidine incorporation. At 12 to 24 hours after compression, there was a 3.3 +/- 0.5-fold (P<.001 versus control) and 3.0 +/- 0.6-fold (P<.002 versus control) increase for 60% and 80% strain, respectively; at 24 to 36 hours after compression, there was a 1.8 +/- 0.5-fold (P<.05 versus control) and 4.3 +/- 0.8-fold (P<.001 versus control) increase. Also, serum-free media conditioned by transiently compressed gel cultures induced DNA synthesis in control, unstimulated smooth muscle cell cultures, suggesting the release of <em>growth</em> <em>factors</em> by transient compression. Although neutralizing antibodies against platelet-derived <em>growth</em> <em>factor</em> did not affect the mechanical induction of DNA synthesis, a neutralizing monoclonal antibody against <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) decreased this induction by 89% and completely blocked the increase in DNA synthesis caused by media conditioned by transiently compressed gels. Media conditioned by transient compression contained elevated levels of FGF-2 (<em>17</em> +/- 5 versus 2 +/- 2 pg/mL for control, P<.005) with no increase in lactate dehydrogenase activity, suggesting release of FGF-2 with sublethal cellular injury.

CONCLUSIONS

A single transient mechanical stimulus increases DNA synthesis in human vascular smooth muscle cells, in part by autocrine or paracrine FGF-2 release.

OBJECTIVE

Activating mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are responsible for several craniosynostosis and chondrodysplasia syndromes as well as some human cancers, including bladder and cervical carcinoma. Despite a high frequency in some benign skin disorders, FGFR3 mutations have not been reported in cutaneous malignancies. Actinic cheilitis (AC) is a sun-induced premalignancy affecting the lower lip that frequently progresses to squamous cell carcinoma (SCC). The objective of this study was to determine if FGFR3 gene mutations are present in AC and SCC of the lip.

METHODS

DNA was extracted and purified from microdissected, formalin-fixed, paraffin-embedded tissue sections of 20 cases of AC and SCC arising in AC. Exons 7, 15, and 17 were PCR amplified and direct sequenced.

RESULTS

Four novel somatic mutations in the FGFR3 gene were identified: exon 7 mutation 742C->>T (amino acid change R248C), exon 15 mutations 1850A->>G (D617G) and 1888G->>A (V630M), and exon 17 mutation 2056G->>A (E686K). Grade of dysplasia did not correlate with presence of mutations.

CONCLUSIONS

The frequency of FGFR3 receptor mutations suggests a functional role for the FGFR3 receptor in the development of epithelial disorders, and perhaps this change may contribute to the pathogenesis of some AC and SCC.

OBJECTIVE

It has been shown that early detection of breast cancer could save lives. Recently, there has been increasing interest in nipple fluid as a potential supplemental avenue for breast cancer diagnosis.

METHODS

In this study, we determined the levels of an angiogenic factor basic fibroblast growth factor (bFGF) in the nipple fluid of healthy subjects as well as patients with benign breast conditions, those at high risk for breast cancer, and patients with active breast cancer. ELISAs were used to measure bFGF.

RESULTS

Nipple fluid bFGF levels were as follows (mean +/- SE): 158 +/- 17 pg/mL from benign breasts, 561 +/- 277 pg/mL from high-risk breasts, and 1,343 +/- 441 pg/mL from cancerous breasts. One-way ANOVA showed that the bFGF levels from cancerous breasts were significantly higher than those from benign and high-risk breasts (P = 0.0001 and P = 0.0193, respectively). After logarithmic transformation was applied to the data, high-risk breast bFGF levels were higher than those from benign breasts (P = 0.0028). With a cutoff level of 250 pg/mL, the sensitivity was 79.2%, specificity was 82.5%, and correct diagnosis was 66.4%. The area under the receiver operating characteristic curve was 0.86.

CONCLUSIONS

We conclude that nipple fluid bFGF levels are progressively elevated in high-risk and cancerous breasts compared with benign breasts. The sensitivity and specificity of this test are promising compared with current breast cancer screening methods, and this test deserves further studies with larger clinical trials. Potential areas of usefulness include the detection of breast cancer risk or breast cancer, as well as the monitoring and/or prediction of the antiangiogenic effect of preventive therapies.

Tumor cells of all types and species tested have been found to produce, in culture, substances that depress the expression of cell-mediated immunity, in the form of delayed-type hypersensitivity reactions in mouse feet. The <em>factors</em> responsible appear related immunologically to the retroviral envelope protein p15E. We have measured the effects of tumor products and conjugates of a p15E-related peptide, CKS-<em>17</em>, on interleukin-2 (IL-2) production by cultured, mitogen-stimulated EL4 cells; in this system IL-2 production is independent of IL-1. Supernatants of cultures of mouse, human and guinea-pig tumor cells inhibited IL-2 production in a dose-dependent fashion. CKS-<em>17</em> conjugates, but not control conjugates, also inhibited IL-2 production. Responses to IL-2 of the CTLL line used were less inhibited by tumor products and very slightly inhibited by CKS-<em>17</em> conjugates. IL-2 receptor density, assayed by flow cytometry, was not inhibited. IL-2 production was inhibited whether the tumor products or CKS-<em>17</em> conjugates were added early or late in the course of culture of stimulated EL4 cells. Inhibition by CKS-<em>17</em> conjugates was selective in that IL-2 production was inhibited to a greater degree than general protein synthesis in EL4 cells, and general protein synthesis by <em>fibroblasts</em> was unaffected. Measurement of IL-2 mRNA suggested that inhibition of IL-2 production was mediated post-transcriptionally. Fractionation of six different tumor supernatants on Sephacryl S-300 revealed a single peak of activity with an apparent molecular mass of 18 kDa. Antibodies to CKS-<em>17</em> conjugates neutralized the inhibitory effect of native tumor products on IL-2 production. Inhibition of IL-2 production, by <em>factors</em> related to p15E, provides a strategically effective means of subversion of host defenses by tumors, and abrogation of this inhibition by means of antibodies might promote host resistance to tumor <em>growth</em>.

BACKGROUND

The activation of oncogenes is an important step in tumorigenesis, and recently, oncogene-induced senescence (OIS) was proposed as a critical barrier against malignant transformation in normal primary cells.

OBJECTIVE

The aim of this study was to examine the activation of fibroblast growth factor receptor 3 (FGFR3) as an oncogene product and OIS in human skin tumours.

METHODS

We investigated the activation of FGFR3 and OIS by mutation and immunohistochemical analysis in skin tumours, including seborrhoeic keratosis, actinic keratosis (AK), Bowen's disease (BD), basal cell carcinoma (BCC) and squamous cell carcinoma (SCC).

RESULTS

Activated point mutations of FGFR3 were identified in four of 22 cases (18%) of seborrhoeic keratosis, but no mutation was detected in the other skin tumours. Twenty-seven of 31 cases (87%) of seborrhoeic keratosis showed moderately to strongly positive expression of the FGFR3 protein, but almost all the other skin tumours were negative. On the other hand, almost all the seborrhoeic keratoses showed negative immunoreactivity for antiphoshohistone H2AX (gamma-H2AX) as a marker of OIS, but 17 of 22 cases (77%) of AK were moderately to strongly positive. Immunoreactivity for gamma-H2AX was significantly greater in AK than in seborrhoeic keratosis, BD, BCC and SCC.

CONCLUSIONS

The activation of FGFR3 might be a common feature in the tumorigenesis in seborrhoeic keratosis, although the activation does not induce a typical oncogenic signal in keratinocytes. In addition, OIS due to some oncogenic signals rather than activation of FGFR3 might be involved in the early skin carcinogenesis related to chronic ultraviolet radiation exposure.

As papillary thyroid carcinoma cells grow surrounding finger-like structures of stromal tissue, we postulated they may secrete a <em>growth</em> <em>factor</em>(s) for mesenchymal cells and that these would be distinct from any mitogenic <em>factors</em> elaborated by follicular carcinomas. Conditioned medium from both the human papillary carcinoma cell line NPA and the follicular carcinoma cell line WRO evoked a 20- to 30-fold increase in [3H]thymidine incorporation into NIH3T3 cell DNA. NPA cell <em>growth</em> <em>factor</em> activity largely eluted with 0.5 mol/L NaCl from a heparin-Sepharose column. NPA-conditioned medium competed in a platelet-derived <em>growth</em> <em>factor</em>-B (PDGF-B) RRA, and the mitogenic activity was partially blocked by an anti-PDGF-BB antibody. An immunoprecipitated PDGF-B-like protein from NPA cells was about <em>17</em> kilodaltons in a reducing gel, but, in contrast to wild-type PDGF-BB, did not change its electrophoretic mobility in an unreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NPA cells expressed an abundant 1.4-kilobase RNA that hybridized to probes for the 5'-untranslated and amino-terminal domains of PDGF-B and was distinct from the 4.2-kilobase wild-type PDGF-B chain transcript. There were no structural changes in the PDGF-B gene, as determined by cytogenetic analysis and restriction mapping. However, the PDGF-B gene in the NPA cells was hypomethylated compared to that in normal thyroid tissue or WRO cells. In contrast, the mitogenic activity of WRO cells bound to heparin with high affinity and was blocked by a basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) antibody. WRO cells contained abundant bFGF mRNA. Both cell lines abundantly expressed transforming <em>growth</em> <em>factor</em>-beta mRNA. Thus, NPA and WRO cells express powerful, yet distinct, mesenchymal cell <em>growth</em> <em>factors</em>. Whereas WRO cells express abundant bFGF, NPA cells produce a novel PDGF-B-like protein, which may correspond to a mutated form of PDGF-B-chain.

OBJECTIVE

To quantify the concentrations of basic fibroblast growth factor (bFGF) and hyaluronan (HA) in the aqueous humor of patients with the exfoliation syndrome (XFS) or exfoliative glaucoma (XFG).

METHODS

Aqueous humor bFGF and HA levels were measured in 13 patients with XFS and in 7 patients with XFG. The results were compared with those obtained from 17 healthy controls.

RESULTS

Mean bFGF levels were significantly higher in the XFG patients than those in the XFS patients, which in turn were higher than the bFGF levels in the healthy individuals. Aqueous humor HA levels in both patients with the XFS and the XFG were significantly higher compared to the controls.

CONCLUSIONS

We suggest that bFGF plays an important role in the pathogenesis of XFS and XFG, as well as in the synthesis of secreted HA, which may result in connective tissue degradation that affects the ocular anterior segment.

Previous studies have provided suggestive evidence for an interaction between ras activation and signalling pathways involved in agonist-stimulated arachidonic acid release in a variety of cell systems. In order to clarify this interaction, we have measured epidermal <em>growth</em> <em>factor</em> (EGF)-stimulated arachidonic acid release in rat-1 <em>fibroblasts</em> transfected with the N-<em>17</em> dominant negative mutation of ras. Cells transfected with the N-<em>17</em> ras mutant, display a markedly attenuated arachidonic acid-release response to EGF, compared to sham-transfected and non-transfected cells. In contrast, the response to phorbol myristate acetate (PMA) was not attenuated in the N-<em>17</em>-mutant expressing cells. No differences were detected between sham-transfected and N-<em>17</em> mutant expressing cells in levels of immunodetectable EGF receptor, cytosolic phospholipase A2 or mitogen-activated protein (MAP) kinase. Attenuation of EGF-stimulated arachidonic acid release in the N-<em>17</em> mutant expressing cells, was accompanied by a marked diminution in EGF-stimulated tyrosine phosphorylation of MAP kinase. We conclude that the signalling pathway involved in epidermal <em>growth</em> <em>factor</em>-stimulated arachidonic acid release is similar to the signalling pathway for mitogenic responses to epidermal <em>growth</em> <em>factor</em> and requires ras activation, likely followed by a downstream cascade of kinases eventuating in MAP kinase activation.

The binding of 125I-labelled epidermal <em>growth</em> <em>factor</em> (EGF) was utilized to monitor possible cell surface effects of polycyclic aromatic hydrocarbon carcinogens. Exposure of confluent C3H 10T1/2 mouse <em>fibroblasts</em> to 1 muM benzo[a]pyrene led to a time-dependent decrease of EGF binding. By 24 h, EGF binding was only 5% that of control cultures. In contrast, benzo[a]pyrene-7,8-diol-9,10-oxide did not significantly alter EGF binding, indicating that the inhibition by benzo[a]pyrene was not simply due to DNA damage. A curvilinear Scatchard plot in the control cells was consistent with the presence of two classes of EGF receptors having differing affinities. Our results suggest that the major effect of benzo[a]pyrene was a reduction in receptor number rather than affinity, although other interpretations have not been excluded. Progesterone, <em>17</em> beta-estradiol, benzo[e]pyrene, cholesterol, phenobarbital, 1,1-bis-(p-chlorophenyl)-2,2,2-trichloroethane, hexachlorobenzene or pregnenolone-16 alpha-carbonitrile, did not inhibit EGF binding. On the other hand, several known inducers of P1-450 were very effective inhibitors of EGF binding. These included: dimethylbenz[a]anthracene, 3-methylcholanthrene, benzo[a]pyrene, benz[a]anthracene, beta-naphthoflavone and alpha-naphthoflavone. We postulate that the binding of certain polycyclic aromatic hydrocarbons to the Ah receptor may induce not only specific drug metabolizing enzymes but also inhibition of EGF binding, and possible other cell effects. Further studies are required to verify this hypothesis.

Expression of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> cDNA or dominantly acting oncogenes, e.g., E1A, in immortalized mouse melanocytes leads to autonomous <em>growth</em> in vitro, depigmentation, and in the case of the oncogenes, tumorigenesis. Because downregulation of pigmentation is a common event in human metastatic melanoma cells grown in culture, we determined the molecular basis of depigmentation in a mouse melanocyte model system. We tested the effect of E1A mutants deficient in their ability to neutralize several regulatory proteins and determined changes in melanogenic gene expression. We identified Microphthalmia as the affected, downregulated transcription <em>factor</em> in melanocytes rendered amelanotic by E1A, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, or the oncogenes ras or neu, and in an amelanotic cell variant of Cloudman S91 mouse melanoma. Against expectations, sequestration of p300, a transcriptional adaptor that mediates responses to cyclic adenosine monophosphate, was not required for the full transforming effects of E1A. Our results suggest that in addition to controlling tyrosinase (albino locus) and tyrosinase-related protein 1 (TR-P1/gp75/brown locus), both known to possess the DNA consensus site for binding the Microphthalmia protein, this transcription <em>factor</em> also controls other melanocyte-specific genes such as pink-eyed dilution and Pmel <em>17</em> (silver), but not tyrosinase-related protein 2 (slaty locus). Furthermore, these findings show that microphthalmia is downregulated not only by experimentally introduced dominantly acting oncogenes but also by the aberrant expression of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and by spontaneous tumorigenic transformation.