TRAF4 overexpression is a common characteristic of human carcinomas.

Tumor necrosis factor receptor (TNFR) associated factor 4 (TRAF4) was initially identified as a gene amplified and overexpressed in breast carcinomas. Our aim was to evaluate whether TRAF4 protein overexpression exists in other cancer types. Immunohistochemistry analysis of tumor samples from 623 patients with 20 different tumor types showed that TRAF4 was overexpressed in 268 tumors (43%), including 82 of 137 lung adenocarcinomas (60%). Interestingly, 32 primary tumors and their matching metastases exhibited mostly similar TRAF4 expression pattern. TRAF4 protein overexpression was limited to cancer cells and the subcellular localization was consistently cytoplasmic in a large majority of cases. To investigate changes in TRAF4 gene copy number, 125 cases from six different types of carcinomas were also analysed by fluorescence in situ hybridization. Out of the 28 cases (22%) showing an increased TRAF4 gene copy number, 23 (82%) were overexpressing the protein. Thus, TRAF4 gene amplification is one of the mechanisms responsible for TRAF4 protein overexpression in human cancers. Considering that TRAF4 is located at 17q11.2 in a region of amplification devoid of known oncogenes and is commonly overexpressed in cancer, our data support an oncogenic role for TRAF4.

Transcription regulation and protein subcellular localization of the truncated basic hair keratin hHb1-DeltaN in human breast cancer cells.

An aberrant truncated hHb1 hair keratin transcript, named hHb1-DeltaN, was previously identified in breast carcinomas. No normal tissue tested so far, including hairy skin, expressed hHb1-DeltaN, indicating that hHb1-DeltaN is related to carcinogenesis. In the present study, we investigated the mechanism by which such truncated transcript was generated in breast cancer cell lines. We found that hHb1-DeltaN transcription is initiated at an unusual cryptic promoter within the fourth intron of the hHb1 gene and is dependent on two proximal Sp1 binding sites for its baseline activity. Moreover, hHb1-DeltaN transcription is increased in response to DNA demethylation by the 5-aza-2'-deoxycytidine drug. This induction is dependent on protein neosynthesis, indicating that an additional factor is required. In addition, we showed that the hHb1-DeltaN transcript is translated in vivo as a truncated hHb1 protein that is missing the 270 amino-terminal residues. The hHb1-DeltaN protein exhibits a filament pattern throughout the cytoplasm and partially co-localizes with cytokeratin filaments, indicating its participation in the cytoskeleton network. hHb1-DeltaN might alter the adhesive properties of cancer cells.

Expression of a truncated form of hHb1 hair keratin in human breast carcinomas.

Human hHb1 belongs to the type II hard keratin family and is physiologically expressed in hair shafts. In the present study, using specific 3' and 5' probes for hHb1, we established that breast carcinomas ectopically express a hHb1 5'-truncated mRNA, and that this transcript is restricted to malignant epithelial cells. Furthermore, an in vitro study indicated that it could be translated. We concluded that, in breast carcinomas, expression of truncated hHb1 is related to epithelial cell transformation. Because the hHb1 gene maps to 12q11-q13, a chromosome region known to present several breakpoints in solid tumours, we propose that the hHb1 gene might represent a target for such alterations.

Lasp-1, a novel type of actin-binding protein accumulating in cell membrane extensions.

The Lasp-1 gene, which has been localized to the q12-q21 region of human chromosome 17, is amplified and overexpressed in human breast cancers. In addition to the previously reported LIM and SH3 domains of Lasp-1, we report here the identification of an actin-binding domain in the core of the protein. This domain is functional as we demonstrate that Lasp-1 binds actin in vivo and in vitro. In addition, confocal analysis of the Lasp-1 subcellular distribution shows that the protein is colocalized with actin at peripheral cell extensions in individual epithelial cancer cells and in transformed fibroblastic cells. Moreover, Lasp-1 is tyrosine phosphorylated in fibroblast cell lines transformed by a constitutively active form of c-Src (c-SrcY527F). Altogether, our results show that Lasp-1 defines a new type of actin-binding protein and suggest that the protein may play a role in a signaling pathway involved in the organization of the cytoskeleton.

Tumor necrosis factor receptor associated factor 4 (TRAF4) expression pattern during mouse development.

This is the first in situ hybridization analysis of expression of a tumor necrosis factor (TNF) receptor associated factor (TRAF) during development. TRAF4 is observed throughout mouse embryogenesis, most notably during ontogenesis of the central (CNS) and peripheral (PNS) nervous system, and of nervous tissues of sensory organs. TRAF4 is preferentially expressed by post-mitotic undifferentiated neurons. Interestingly, TRAF4 remains expressed in the adult hippocampus and olfactory bulb, known to contain multipotential cells responsible for neoneurogenesis.

Tumor necrosis factor (TNF)-mediated kinase cascades: bifurcation of nuclear factor-kappaB and c-jun N-terminal kinase (JNK/SAPK) pathways at TNF receptor-associated factor 2.

TNF-induced activation of the transcription factor NF-kappaB and the c-jun N-terminal kinase (JNK/SAPK) requires TNF receptor-associated factor 2 (TRAF2). The NF-kappaB-inducing kinase (NIK) associates with TRAF2 and mediates TNF activation of NF-kappaB. Herein we show that NIK interacts with additional members of the TRAF family and that this interaction requires the conserved "WKI" motif within the TRAF domain. We also investigated the role of NIK in JNK activation by TNF. Whereas overexpression of NIK potently induced NF-kappaB activation, it failed to stimulate JNK activation. A kinase-inactive mutant of NIK was a dominant negative inhibitor of NF-kappaB activation but did not suppress TNF- or TRAF2-induced JNK activation. Thus, TRAF2 is the bifurcation point of two kinase cascades leading to activation of NF-kappaB and JNK, respectively.

Identification and characterization of an IkappaB kinase.

Activation of the transcription factor NF-kappaB by tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the NF-kappaB-inducing kinase (NIK). In a yeast two-hybrid screen for NIK-interacting proteins, we have identified a protein kinase previously known as CHUK. Overexpression of CHUK activates a NF-kappaB-dependent reporter gene. A catalytically inactive mutant of CHUK is a dominant-negative inhibitor of TNF-, IL-1-, TRAF-, and NIK-induced NF-kappaB activation. CHUK associates with the NF-kappaB inhibitory protein, IkappaB-alpha, in mammalian cells. CHUK specifically phosphorylates IkappaB-alpha on both serine 32 and serine 36, modifications that are required for targeted degradation of IkappaB-alpha via the ubiquitin-proteasome pathway. This phosphorylation of IkappaB-alpha is greatly enhanced by NIK costimulation. Thus, CHUK is a NIK-activated IkappaB-alpha kinase that links TNF- and IL-1-induced kinase cascades to NF-kappaB activation.

Expression pattern of human hair keratin basic 1 (hHb1) in hair follicle and pilomatricoma.

Using in situ hybridization, human hair keratin basic 1 (hHb1) gene expression was investigated in human normal scalp. hHb1 transcripts were specifically detected in the cortical cells of hair shaft but neither in the outer and inner root sheaths, nor in the hair cuticle or the medulla. hHb1 expression was detected strongly in cortical cells located from the beginning of the keratogenous zone up to the isthmus. These data specify the localization of hHb1 expression. Furthermore, neoplasms with follicular differentiation, including trichoblastoma, trichoepithelioma, pilomatricoma, pilar carcinoma and basal-cell carcinoma, were analysed for hHb1 gene expression. One of the 4 pilomatricoma specimens examined exhibited a very high level of hHb1 transcripts. Interestingly, this labeling was specifically associated to a transitional cell layer en route to trichocytic differentiation, providing evidence that in pilomatricoma, epithelial germ cells can differentiate towards hair shaft keratinocytes before evolving in ghost cells.

MLN64 exhibits homology with the steroidogenic acute regulatory protein (STAR) and is over-expressed in human breast carcinomas.

The MLN64 gene, which is localized in q12-q21 of the human chromosome 17, encodes a novel protein containing 2 distinct domains. At the N-terminal, MLN64 exhibits a potential trans-membrane region, while at the C-terminal, it shares homology with the F26F4.4 protein of Coenorhabditis elegans and the steroidogenic acute regulatory (StAR) protein, a mitochondrial protein which is involved in steroid-hormone synthesis. By comparing the C-terminal part of these proteins, we defined a novel protein domain, which we termed SHD for "StAR Homology Domain". Of the 93 primary invasive breast carcinomas that were examined, 14 were found to over-express MLN64. These 14 tumors also expressed high c-erbB-2 transcript levels, which were not detected in the MLN64-negative tumors. MLN64 mRNA and protein were specifically detected in malignant cells of breast carcinomas. MLN64 protein was localized within bundle-like structures distributed throughout the cell cytoplasm and condensed in a perinuclear patch, suggesting an association with a specific cell compartment. When the N-terminal part of MLN64 was deleted, MLN64 was uniformly distributed in the cell cytoplasm, indicating that N-terminal part is involved in the specific cytoplasmic localization of MLN64. The homology between the C-terminal part of MLN64 and the functional StAR domain (SHD) suggests that MLN64 and StAR, although distributed in different cellular compartments, may both play a role in steroidogenesis. In this case, the high levels of MLN64 observed in some breast carcinomas could contribute to the progression of these tumors through increased intratumoral steroidogenesis.

Two distinct amplified regions at 17q11-q21 involved in human primary breast cancer.

Chromosomal segment 17q11-q21 is a commonly amplified region in human breast carcinomas. Several lines of evidence suggest that ERBB2 is the gene responsible for the emergence of this amplicon, but four novel genes (called MLN 50, MLN 51, MLN 62, and MLN 64) in 17q11-q21 have recently been found to be amplified and overexpressed in breast cancer cell lines. We investigated 98 primary breast tumors for amplification of these five loci. Twenty-five tumors (25.5%) showed amplification of at least one of these markers, but most amplifications did not encompass all of the tested loci. The genes most frequently amplified were ERBB2 and MLN 64 (22 of 25 amplified cases). MLN 64 was always coamplified with ERBB2, and to a similar level. Amplification of these five genes always leads to overexpression of their mRNA; we observed no cases of overexpression without amplification in any of these genes. Our results suggest that: (a) an independent, amplified region defined by MLN 62 (also called CART1 or TRAF4) is located in 17q11-q12; (b) in addition to ERBB2, MLN 64 is a major target for the 17q12-q21 amplicon; and (c) these MLN genes could be of pathogenetic significance in breast cancer.

Stromelysin-3 in the biology of the normal and neoplastic mammary gland.

Stromelysin-3 (ST3) is an extracellular proteinase predominantly expressed in fibroblasts. The particular structural features and in vitro functions of this molecule suggest it could be the first member of a new subgroup of the matrix metalloproteinase family. ST3 is transiently expressed during mammary gland post-weaning involution, embryonic implantation, various organogeneses, and during amphibian metamorphosis. Moreover, ST3 is expressed in a panel of human invasive carcinomas including breast, colon, and head and neck carcinomas. Almost all ST3-expressing tissues show intense extracellular matrix remodeling activities including the loss of basement membrane integrity. Thus, either directly, or indirectly in association with other proteinases, ST3 might be involved in tissue remodeling processes occurring in both physiological and pathological processes. In vitro and in vivo studies using malignant cells stably transfected in such a way as to modulate their ST3 expression levels indicate that ST3 modifies neither cell proliferation nor invasive properties, but rather favors tumor cell survival in host tissues. This hypothesis is consistent with clinical data showing that ST3 expression could be predictive of tumor progression leading to metastases.

Lasp-1 (MLN 50) defines a new LIM protein subfamily characterized by the association of LIM and SH3 domains.

MLN 50 was previously identified in a cDNA library of breast cancer metastasis. In this study, we show that MLN 50, which is expressed at a basal level in normal tissues, is overexpressed in 8% of human breast carcinomas most often together with c-erbB-2. MLN 50 cDNA encodes a putative protein of 261 residues, named Lasp-1 (LIM and SH3 protein) since it contains a LIM motif and a domain of Src homology region 3 (SH3) at the amino- and the C-terminal parts of the protein, respectively. Thus, Lasp-1 defines a new LIM protein subfamily.

Comparative expression of the psoriasin (S100A7) and S100C genes in breast carcinoma and co-localization to human chromosome 1q21-q22.

Using differential screening of a breast cancer cDNA library, we isolated a cDNA encoding the psoriasin (S100A7) protein, previously identified in psoriatic epidermis. In the present study, we demonstrate that the psoriasin gene is expressed in breast cancer cell lines and in cancer cells of some breast carcinomas but not in any non-cancerous tissues examined, except skin. Another S100 gene, S100C, which we co-localized with the psoriasin gene to human chromosome 1q21-q22, was found to be expressed in most tissues and cell lines evaluated. These findings add support to the concept that the S100 genes clustered in human chromosome 1q21-q22 are individually controlled and that some of them may be involved in the regulation of cell transformation and/or differentiation.

Presence of a new conserved domain in CART1, a novel member of the tumor necrosis factor receptor-associated protein family, which is expressed in breast carcinoma.

CART1, a novel human gene, encodes a putative protein exhibiting three main structural domains: first, a cysteine-rich domain located at the amino-terminal part of the protein, which corresponds to an unusual RING finger motif; second, an original cysteine-rich domain located at the core of the protein and constituted by three repeats of an HC3HC3 consensus motif that we designated the CART motif, and which might interact with nucleic acid; third, the carboxyl-terminal part of the CART1 protein corresponds to a TRAF domain known to be involved in protein-protein interactions. Similar association of RING, CART, and TRAF domain was observed in the human CD40-binding protein and in the mouse tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), both involved in signal transduction mediated by the TNF receptor family and in the developmentally regulated Dictyostelium discoideum DG17 protein. CART1 is specifically expressed by epithelial cells in breast carcinomas and metastases. Moreover, in these malignant cells, the CART1 protein is localized in the nucleus. Altogether, these observations indicate that CART1 may be involved in TNF-related cytokine signal transduction in breast carcinoma.

Effect of vesical outlet obstruction on detrusor contractility and passive properties in rabbits.

Outlet obstruction was induced in 16 New Zealand rabbits by implanting a polyethylene tube (20 F) for a period of three months. The tube was slit longitudinally and placed around the bladder neck, between the ureters and vasa deferentia. It was left open to induce moderate obstruction and closed by a suture to induce severe obstruction. The animals were studied by cutting consecutive rings from each bladder, which were subjected to the following studies: morphology, contractility and mechanical properties. Morphology. Histology sections of the rings, studied by Hematoxylin and Eosin and Masson-Trichrome stains, demonstrated smooth muscle hypertrophy in moderate obstruction, while hyperplasia was the predominant response in severe obstruction. The average nuclear count per square millimeter of smooth muscle was 251 in controls, 87 in moderate obstruction and 705 in severe obstruction. Bladder wall thickness was significantly increased after both moderate and severe obstruction, as compared to controls. Contractility and mechanical properties. Each ring was tested in a muscle chamber under conditions of maximal electrical stimulation (100 V, 2 ms, 40 Hz). For each ring, full force-length relationships (active and passive) were obtained by stretching the rings in successive increments until length of maximal active force development was reached. Force was normalized per unit cross-sectional area (stress), and length according to a "reference" underformed state (per cent of unloaded length). The maximum active stress of the rings was taken as a measure of bladder contractility, and the rate of increase in passive force as a measure of detrusor stiffness. In all groups, the body of the detrusor exerted better contractility, as compared to a rigid, less contractile base. In the obstructed groups, detrusor contractility was significantly decreased at the body level, with increased stiffness, as compared to controls. The length at which maximum contractility was exerted, however, increased in moderate obstruction, and decreased in severe obstruction.

A new catheter to measure urethral compliance in females: normal values.

We describe a new probe made of several F5 Vinyl tubes glued and staggered together in such a way that the catheter progressively increases in size (from F5 to F30), as more tubes are joined. At each size, pressure is recorded through a single side hole in one of the tubes. Pressure within the urethra is thus successively recorded from F5 to F30 as the catheter is further introduced. A single pressure transducer is used since the lines of the probe are separately and successively connected to the transducer through a 6-channel manifold, as one proceeds to the next diameter. Pressure diameter curves are obtained and reported on a graph. The data are analyzed by fitting the graphs with a one exponential equation P = PoeaD; 2 parameters are computed: Po, the initial pressure when the urethra is catheter free, and a, the rate of exponential increase in pressure as higher diameters are introduced to measure urethral wall stiffness. This catheter was used to measure the maximum urethral pressure in 10 young nulliparous volunteers, at each level of stretch. The values of Po and a were found to be respectively 74.4 +/- 7.7 cm. H2O and 0.08 +/- 0.02. The quantitative measurement of urethral compliance constitutes for us an integral part in the urodynamics assessment of female patients presenting with lower urinary tract dysfunction.

Abnormal urethral compliance in females diagnosis, results and treatment. Preliminary study.

We measured urethral compliance in 57 patients using a catheter of increasing diameter. The measurement makes it possible to differentiate clearly between rigidity and hyperlaxity of the female urethra. In both instances, the initial pressure (Po) is lower than normal, but the rate of pressure increase (a), with larger catheter sizes, is significantly higher in cases of urethral rigidity and on the contrary parallel to normal in cases of hyperlaxity. This distinction allows for more accuracy in the determination of therapeutic indication. An inversely proportional correlation was found between the initial urethral pressure and the rate of pressure increase (r = 0.74). The initial pressure is generally lower in older women (r = 0.56).

Effect of diethylstilbestrol on bladder contractility in male rats.

The effect of diethylstilbestrol (DES) on bladder contractility was studied in vitro in 8 rats while 5 untreated animals were used as normal controls. Of the treated animals, 4 received 0.05 mg./day of DES during 8 weeks while another 4 were treated for 14 weeks with the same dose. We studied the animals by cutting consecutive rings from each bladder and testing them in a muscle chamber, under conditions of maximal electrical stimulation which had been previously determined in 3 animals. Parameters that produced maximal contractions were: 100 V, 40 Hz, and 2 msec. For each ring, we obtained full force-length relationships by stretching the rings in successive increments until length of maximal active force development was reached. For each bladder, alternate rings were tested in the muscle chambers while the remaining rings were used to determine collagen percentage with Woessner assay. In all animals, the ring with the largest diameter which was obtained from the widest portion of the bladder gave the highest contraction. The maximal active force of this ring was taken as a measure of contractility of the bladder. For controls, the maximal active force was 7.30 +/- 0.3 g. After 8 weeks of DES treatment, it decreased to 5.9 +/- 0.79 g and after 14 weeks, it further decreased to 2.15 +/- 0.99 g. The reduction in active force from controls after 8 and 14 weeks were statistically significant. Passive force and collagen content, however, did not change after either 8 or 14 weeks. In conclusion, these data show that DES decreases bladder contractility in male rats without affecting passive forces and muscle-collagen ratio.

The elastic behavior of the urinary bladder for large deformations.

The purpose of this paper is to investigate the theoretical basis for the pressure-distension behavior of the urinary bladder. A finite strain theory is developed for hollow spherical structures and it is shown that the Treloar model is a good prototype only for rubber balloons. The pressure-extension ratio relationship is inverted to lead a general form of strain energy function, and fitted by an empirical relation involving one exponential. The following form of strain energy function is derived: W(lambda, lambda, lambda -2) = C1 (P(1), a) + P(1)C2 (a, lambda)ea(lambda -1). Where C1(P(1), a) is a constant (N m-2), P(1) is the initial pressure, a is the rate of pressure increase and C2 (a, lambda) a third degree polynomial relation. P(1) and a are experimentally determined through volumetric pressure-distension data. It is verified that this type of energy function is also valid for uniaxial loading experiments by testing strips coming from the same bladder for which P(1) and a were computed. There is a good agreement between the experimental points and the theoretical stress-strain relation. Finally, the strain energy function is plotted as a function of the first strain invariant and appears to be of an exponential nature.