Incomplete penetrance for isolated congenital asplenia in humans with mutations in translated and untranslated RPSA exons.

Isolated congenital asplenia (ICA) is the only known human developmental defect exclusively affecting a lymphoid organ. In 2013, we showed that private deleterious mutations in the protein-coding region of RPSA, encoding ribosomal protein SA, caused ICA by haploinsufficiency with complete penetrance. We reported seven heterozygous protein-coding mutations in 8 of the 23 kindreds studied, including 6 of the 8 multiplex kindreds. We have since enrolled 33 new kindreds, 5 of which are multiplex. We describe here 11 new heterozygous ICA-causing RPSA protein-coding mutations, and the first two mutations in the 5'-UTR of this gene, which disrupt mRNA splicing. Overall, 40 of the 73 ICA patients (55%) and 23 of the 56 kindreds (41%) carry mutations located in translated or untranslated exons of RPSA. Eleven of the 43 kindreds affected by sporadic disease (26%) carry RPSA mutations, whereas 12 of the 13 multiplex kindreds (92%) carry RPSA mutations. We also report that 6 of 18 (33%) protein-coding mutations and the two (100%) 5'-UTR mutations display incomplete penetrance. Three mutations were identified in two independent kindreds, due to a hotspot or a founder effect. Finally, RPSA ICA-causing mutations were demonstrated to be de novo in 7 of the 23 probands. Mutations in RPSA exons can affect the translated or untranslated regions and can underlie ICA with complete or incomplete penetrance.

Cloning and expression analysis of the 37-kDa laminin receptor precursor gene from Hyriopsis cumingii.

Hyriopsis cumingii is an economically important freshwater pearl mussel with high pearl quality that is endemic in China. Investigation of genes relevant to shell formation is important for increased pearl output. The substances that form mollusk shells are secreted by epithelial cells in the mantle, the proliferation of which influences secretion ability. This study focused on the proliferation-related 37-kDa laminin receptor precursor (37LRP) of H. cumingii. The full-length cDNA (1133 bp) encoding this 300-amino acid protein was cloned from the mantle. Quantitative fluorescence analysis showed that 37LRP expressed in eight tissues, with the highest expression observed in the liver, and its expression pattern in the mantle reflected shell repair. During repair, 37LRP expression was higher in the experimental shell repair group than that in the control group, exhibiting an initial increase followed by a decrease in expression, and returning to basal levels on completion of the repair. A similar trend was also observed with respect to immunity and cellular metabolism. Expression of the 37LRP protein in the experimental group was significantly higher than that in the control group at the first and second days after shell injury. After 4 days, 37LRP expression in the experimental group was lower than that in the control group. In situ hybridization revealed a strong positive signal corresponding to the 37LRP mRNA at the horny grooves of the mantle, evagination, and in epithelial cells of the velum, which implicated these areas in the repair and formation of the cuticle, prismatic layer, and nacre.

The unusual response of serotonergic neurons after CNS Injury: lack of axonal dieback and enhanced sprouting within the inhibitory environment of the glial scar.

Serotonergic neurons possess an enhanced ability to regenerate or sprout after many types of injury. To understand the mechanisms that underlie their unusual properties, we used a combinatorial approach comparing the behavior of serotonergic and cortical axon tips over time in the same injury environment in vivo and to growth-promoting or growth-inhibitory substrates in vitro. After a thermocoagulatory lesion in the rat frontoparietal cortex, callosal axons become dystrophic and die back. Serotonergic axons, however, persist within the lesion edge. At the third week post-injury, 5-HT+ axons sprout robustly. The lesion environment contains both growth-inhibitory chondroitin sulfate proteoglycans (CSPGs) and growth-promoting laminin. Transgenic mouse serotonergic neurons specifically labeled by enhanced yellow fluorescent protein under control of the Pet-1 promoter/enhancer or cortical neurons were cultured on low amounts of laminin with or without relatively high concentrations of the CSPG aggrecan. Serotonergic neurons extended considerably longer neurites than did cortical neurons on low laminin and exhibited a remarkably more active growth cone on low laminin plus aggrecan during time-lapse imaging than did cortical neurons. Chondroitinase ABC treatment of laminin/CSPG substrates resulted in significantly longer serotonergic but not cortical neurite lengths. This increased ability of serotonergic neurons to robustly grow on high amounts of CSPG may be partially due to significantly higher amounts of growth-associated protein-43 and/or ß1 integrin than cortical neurons. Blocking ß1 integrin decreased serotonergic and cortical outgrowth on laminin. Determining the mechanism by which serotonergic fibers persist and sprout after lesion could lead to therapeutic strategies for both stroke and spinal cord injury.

Expression of 67-kDa laminin receptor was associated with tumor progression and poor prognosis in epithelial ovarian cancer.

OBJECTIVE: 67-kDa laminin receptor (67LR) has been identified as a prognostic biomarker for a variety of human cancers. We investigated the clinical significance of 67LR expression and its functional role in epithelial ovarian cancer (EOC). METHODS: 67LR expression was evaluated by immunohistochemistry in 62 patients with EOC. We assessed the correlation of 67LR expression with clinical characteristics. In vitro experiment was performed for 67LR with inhibition using siRNA to evaluate its role in cell survival, apoptosis, and invasion in EOC cells. RESULTS: 67LR was predominantly expressed on the cell membrane in the majority of EOC samples (45/62, 73%). 67LR expression was significantly correlated with advanced stage (P=0.001). Patients with 67LR expression had shorter progression-free survival among all the patients (P=0.010) and in particular among patients with advanced stages (P=0.046). When 67LR expression was inhibited by siRNA in EOC cells (HeyA8 and A2780), there was a significant decrease of cell proliferation and invasion as well as increase of apoptosis. CONCLUSION: These findings suggest that 67LR expression may play an important role in tumor progression into advanced stage with poor prognosis in EOC and down-regulation of 67LR on tumor cells may be a therapeutic target in those patients.

Changes in cell migration-related molecules expressed by thymic microenvironment during experimental Plasmodium berghei infection: consequences on thymocyte development.

We previously showed alterations in the thymus during experimental infection with Plasmodium berghei. Such alterations comprised histological changes, with loss of cortical-medullary limits, and the intrathymic presence of parasites. As the combination of chemokines, adhesion molecules and extracellular matrix (ECM) is critical to appropriate thymocyte development, we analysed the thymic expression of ECM ligands and receptors, as well as chemokines and their respective receptors during the experimental P. berghei infection. Increased expression of ECM components was observed in thymi from infected mice. In contrast, down-regulated surface expression of fibronectin and laminin receptors was observed in thymocytes from these animals. Moreover, in thymi from infected mice there was increased CXCL12 and CXCR4, and a decreased expression of CCL25 and CCR9. An altered thymocyte migration towards ECM elements and chemokines was seen when the thymi from infected mice were analysed. Evaluation of ex vivo migration patterns of CD4/CD8-defined thymocyte subpopulations revealed that double-negative (DN), and CD4(+) and CD8(+) single-positive (SP) cells from P. berghei-infected mice have higher migratory responses compared with controls. Interestingly, increased numbers of DN and SP subpopulations were found in the spleens of infected mice. Overall, we show that the thymic atrophy observed in P. berghei-infected mice is accompanied by thymic microenvironmental changes that comprise altered expression of thymocyte migration-related molecules of the ECM and chemokine protein families, which in turn can alter the thymocyte migration pattern. These thymic disturbances may have consequences for the control of the immune response against this protozoan.

LRP::FLAG Reduces Phosphorylated Tau Levels in Alzheimer's Disease Cell Culture Models.

BACKGROUND: Alzheimer's disease (AD) is characterized by amyloid-ß (Aß) plaque and neurofibrillary tangle formation, respectively. Neurofibrillary tangles form as a result of the intracellular accumulation of hyperphosphorylated tau. Telomerase activity and levels of the human reverse transcriptase (hTERT) subunit of telomerase are significantly decreased in AD. Recently, it has been demonstrated that the 37 kDa/67 kDa laminin receptor (LRP/LR) interacts with telomerase and is implicated in Aß pathology. Since both LRP/LR and telomerase are known to play a role in the Aß facet of AD, we hypothesized that they might also play a role in tauopathy. OBJECTIVE: This study aimed to determine if LRP/LR has a relationship with tau and whether overexpression of LRP::FLAG has an effect on tauopathy-related proteins. METHODS: We employed confocal microscopy and FRET to determine whether LRP/LR and tau co-localize and interact. LRP::FLAG overexpression in HEK-293 and SH-SY5Y cells as well as analysis of tauopathy-related proteins was assessed by western blotting. RESULTS: We demonstrate that LRP/LR co-localizes with tau in the perinuclear cell compartment and confirmed a direct interaction between LRP/LR and tau in HEK-293 cells. Overexpression of LRP::FLAG in HEK-293 and SH-SY5Y cells decreased total and phosphorylated tau levels with a concomitant decrease in PrPc levels, a tauopathy-related protein. LRP::FLAG overexpression also resulted in increased hTERT levels. CONCLUSION: This data suggest that LRP/LR extends its role in AD through a direct interaction with tau, and recommend LRP::FLAG as a possible alternative AD therapeutic via decreasing phosphorylated tau levels.

Susceptibility of colorectal cancer cells to Sindbis virus infection.

Sindbis virus (SIN), a member of the Togaviridae family, infects a broad range of cells and has been shown to be an effective anti-tumor agent. The infection efficiency of the virus, however, varies greatly among target cells. In this report, we compared the ability of SIN to infect colorectal cancer cells and cells of other cancer origin. While tumor cells from breast, leukemia, and prostate cancers were largely resistant to SIN infection, nine of the ten colorectal cancer cell lines tested were sensitive to SIN infection. Moreover, SIN susceptibility correlated with the metastatic potential of the colorectal cancer cells. Two highly aggressive and invasive cell lines, SW620 and COLO-320DM were the most sensitive to SIN infection. Similarly, SIN preferentially targeted metastatic tumor cells in a mouse xenograft model for colon cancer progression. The higher infection rate was not due to increased expression of the 67kD laminin receptor, a specific receptor for SIN infection, although viral attachment and entry were markedly enhanced in SW620 cells. These results suggest that SIN may employ a novel cell attachment/entry mechanism during infection, allowing selective targeting of colorectal cancer cells.

Expression of laminin receptor 1 in gestational trophoblastic diseases and normal placenta and its relationship with the development of postmolar tumors.

OBJECTIVES: To investigate the expression of laminin receptor 1 (LR1), a non-integrin-type laminin receptor, in gestational trophoblastic diseases and normal first-trimester placenta, since it may play a role in controlling trophoblast invasion in normal and molar pregnancies. METHODS: Paraffin sections from 24 gestational age controlled normal first-trimester placentas, 47 partial moles, 56 complete moles, 3 invasive moles, 4 gestational choriocarcinomas, and 1 placental-site trophoblastic tumor were studied immunohistochemically for expression of LR1. RESULTS: In complete and partial moles, decidual cells showed significantly stronger LR1 protein staining compared to the normal placenta (p<0.01). When compared to the partial moles, weak staining in less than 33% of decidual cells was also more prominent in the normal placenta (p<0.05). Complete and partial moles, invasive moles, choriocarcinomas, and placental-site tumors did not differ from each other with respect to staining intensity. Strong immunostaining for LR1 in decidual cells, cytotrophoblasts, syncytiotrophoblasts, and extracellular matrix cells of partial and complete moles was not significantly correlated with the development of persistent postmolar gestational trophoblastic tumors. CONCLUSIONS: LR1 may be important in the pathogenesis of gestational trophoblastic diseases. The increased expression of LR1 in decidual cells of partial and complete moles may not influence the development of persistent gestational trophoblastic tumor. Since they are seen rarely, multicentric studies should be planned to study LR1 expression in invasive moles and gestational trophoblastic neoplasms.

Overexpression of malignancy-associated laminins and laminin receptors by angiotropic human melanoma cells in a chick chorioallantoic membrane model.

BACKGROUND: As distinct from intravascular/lymphatic dissemination, extravascular migratory metastasis (EVMM) has been described as a potential additional mechanism of melanoma spread in which tumor cells migrate along the external surfaces of vessels. Angiotropic melanoma cells are linked to the endothelium by a matrix containing laminin. In addition, it has been shown that C16 laminin-derived peptide increases extravascular migration of human green fluorescent protein (GFP) melanoma cells along vessels in a chicken chorioallantoic membrane model (CAM). In this study, we have tested the hypothesis that expression levels of some genes related to lamimin and metastasis are differentially expressed in vascularized angiotropic melanoma areas vs. avascular melanoma areas from the same tumor. DESIGN: C8161 human melanoma cells in a shell-less chick CAM assay were used to study EVMM associated with the presence of vascularized angiotropic melanoma areas. For both high-quality histomorphology and RNA preservation in paraffin-embedded tissue, we used a methanol-based fixative coupled with microwave-assisted rapid tissue processing as previously described. Using laser capture microdissection, angiotropic melanoma areas as well as avascular areas were microdissected. Using quantitative real time polymerase chain reaction (QRT-PCR), six genes have been studied: LAMC2 (laminin gamma2 chain), LAMA4 (laminin alpha4 chain), ITGB1 (integrin beta1), ITGB3 (integrin beta3), RSPA (ribosomal protein), and MMP2 (matrix metallopeptidase 2). QRT-PCR data were normalized to human GAPDH housekeeping gene and values were compared against Human Total RNA. Final results were expressed as percentage of expression. RESULTS: All tumors demonstrated a similar pattern, i.e. EVMM of angiotropic melanoma cells. The microdissected histopathological sections presented both angiotropic areas and avascular areas. All genes were overexpressed in angiotropic melanoma areas vs. avascular melanoma areas, especially LAMC2, LAMA4 and ITGB3 (respectively, 165.18, 208.86, and 483.69%). CONCLUSION: This study shows that several genes related to laminin are overexpressed in angiotropic melanoma areas vs. avascular melanoma areas. Since extravascular migration of melanoma cells along vessels has been demonstrated in the CAM model, taken together these results suggests that some laminins and laminin receptors may play a role in extravascular migratory metastasis. This model may represent a promising strategy to analyze differential gene expression in EVMM.

Overexpression of laminin receptor 1 on decidual cells in partial and complete mole.

OBJECTIVES: Laminin receptor 1 (LR1), a non-integrin-type laminin receptor, has been described in several tumors and may play a role in tumor invasion. It is well known that LR1 can modify the conformation and degradation of laminin thus enhancing tumor cell invasion. LR1 may play a role in controlling trophoblast invasion in normal and molar pregnancies. MATERIALS AND METHODS: Real-time polymerase chain reaction (RT-PCR) was performed on total cDNA from 17 gestational age-controlled normal placentas, 10 complete moles (CM), and 4 partial moles (PM). Immunolocalization of LR1 was performed on paraffin sections prepared from 17 age-controlled placentas, 17 PM, and 19 CM. RESULTS: RT-PCR demonstrated a 13-fold increase in LR1 mRNA expression in molar tissues (PM and CM combined) versus normal placentas (p=0.012). Immunohistochemical analysis revealed LR1 localized to the decidual cells. In normal placenta LR1 localized to the decidual cell membrane. However, in PM and CM, LR1 was additionally noted in the cytoplasm of decidual cells. Interestingly, with immunohistochemistry method, we found that PM demonstrated higher protein expression of LR1 than either normal placenta or CM (p=0.001 and p=0.024, respectively). CONCLUSION: LR1 is expressed in both the decidual cells of normal placenta and mole (CM and PM). Decidual cells in CM and PM express LR1 significantly greater than the decidual cells in placenta. The underlying mechanism of how molar tissue may be associated with enhanced expression of LR1 in the maternal endometrium is unclear.

Integrins (alpha7beta1) in muscle function and survival. Disrupted expression in merosin-deficient congenital muscular dystrophy.

Mutations in genes coding for dystrophin, for alpha, beta, gamma, and delta-sarcoglycans, or for the alpha2 chain of the basement membrane component merosin (laminin-2/4) cause various forms of muscular dystrophy. Analyses of integrins showed an abnormal expression and localization of alpha7beta1 isoforms in myofibers of merosin-deficient human patients and mice, but not in dystrophin-deficient or sarcoglycan-deficient humans and animals. It was shown previously that skeletal muscle fibers require merosin for survival and function (Vachon, P.H., F. Loechel, H. Xu, U.M. Wewer, and E. Engvall. 1996. J. Cell Biol. 134:1483-1497). Correction of merosin deficiency in vitro through cell transfection with the merosin alpha2 chain restored the normal localization of alpha7beta1D integrins as well as myotube survival. Overexpression of the apoptosis-suppressing molecule Bcl-2 also promoted the survival of merosin-deficient myotubes, but did not restore a normal expression of alpha7beta1D integrins. Blocking of beta1 integrins in normal myotubes induced apoptosis and severely reduced their survival. These findings (a) identify alpha7beta1D integrins as the de facto receptors for merosin in skeletal muscle; (b) indicate a merosin dependence for the accurate expression and membrane localization of alpha7beta1D integrins in myofibers; (c) provide a molecular basis for the critical role of merosin in myofiber survival; and (d) add new insights to the pathogenesis of neuromuscular disorders.

Genes differentially expressed in responsive and refractory acute leukemia.

DNA microarray in 22 patients with acute leukemia revealed genes which were differentially expressed. Ribosomal protein SA (RPSA), minichromosome maintenance deficient 2 (MCM2) and heterogeneous nuclear ribonucleoprotein A1 (HNRPA1) were significantly upregulated (p<0.05, t test) in refractory patients, suggesting that they may play a role in refractoriness in acute leukemia and could be biomarkers of prognosis.

Cloning and characterization of a novel spermiogenesis-related gene, T6441, in rat testis.

We report in the present study the cloning and characterization of a novel gene, named T6441, initially derived by the suppressive subtracted hybridization (SSH) cDNA library. The full-length T6441cDNA was 664 bp long, containing a complete open-reading frame for a protein of 149 amino acids (aa). The protein bears no homology to any reported genes. It is predicted that the molecular mass was about 16.7 kDa. Northern blot analysis showed that the T6441 gene had about 4 transcripts in adult rat testis and was temporally regulated in a stage-dependent manner in the testis. In situ hybridization showed that T6441 mRNA was specifically localized in spermatids, and its expression level varied in the cells at different stages of the testicular development, with the highest level at steps 7-14. RT-PCR results showed that the T6441 mRNA was transcribed in most of the tested tissues with its strongest signal in the testis. Recombinant T6441 protein was prepared, purified, and was used to raise rabbit. Western blot analysis using the antiserum revealed four possible testicular specific proteins with their molecular weights being about 22, 25, 50 and 55 kDa respectively. The T6441 protein was expressed mainly in the cytoplasm of spermatids with the maximal levels at steps 12-19. At step 19 spermatid, the T6441 was mainly localized in the residual bodies. The cytoplasm localization of T6441 protein was supported by transient over expression of GFP-fusion protein in Hela cells. Interestingly, the expression of T6441 caused death of transfected cells within 48 h. Our preliminary experimental results suggest that the T6441 gene may play a role in cytoplasm movement and removal during spermiogenesis.

Characterization of 67 kD laminin receptor, a protein whose gene is overexpressed on treatment of cells with anti-benzo[a]pyrene-7,8-diol-9,10-epoxide.

The molecular mechanisms potentially related to tumorigenesis induced by anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (anti-BPDE) were investigated by suppression subtractive hybridization of the human bronchial epithelial cells (16HBE) carcinoma induced by BPDE-transformed 16HBE cells (16HBE-C). The 67 kD laminin receptor gene (67LR1) is one of the screened overexpressed genes in 16HBE-C cells when compared with 16HBE. In order to understand the main functions of 67LR1 gene, we amplified the full length of 67LR1 gene using reverse transcription-polymerase chain reaction (RT-PCR) method. The amplified gene products were inserted into pcDNA 3.1 Directional TOPO expression vector. We then transfected 16HBE cells with this vector and derived stable transfected 16HBE cell lines containing the 67LR1 gene by using lipofectin and G418 selection protocols. The expression products of transfected genes were analyzed by semiquantitative RT-PCR. Soft agar growth assay was carried out to identify the malignant features of 67LR1 gene. The stable transfected cell lines can form colonies in soft agar. Further, the transfected cells showed morphological changes compared to the control cells, such as the obvious pseudopods. These data suggest that the 67LR1 gene may be related to malignant transformation induced by the anti-BPDE. The 67LR1 protein may be related to the directionality of cell movement.

Using sindbis viral vectors for specific detection and suppression of advanced ovarian cancer in animal models.

We studied the therapeutic value of Sindbis vectors for advanced metastatic ovarian cancer by using two highly reproducible and clinically accurate mouse models: a SCID xenograft model, established by i.p. inoculation of human ES-2 ovarian cancer cells, and a syngenic C57BL/6 model, established by i.p. inoculation of mouse MOSEC ovarian cancer cells. We demonstrate through imaging, histologic, and molecular data that Sindbis vectors systemically and specifically infect/detect and kill metastasized tumors in the peritoneal cavity, leading to significant suppression of the carcinomatosis in both animal models. Use of two different bioluminescent genetic markers for the IVIS Imaging System permitted demonstration, for the first time, of an excellent correlation between vector delivery and metastatic locations in vivo. Sindbis vector infection and growth suppression of murine MOSEC tumor cells indicate that Sindbis tumor specificity is not attributable to a species difference between human tumor and mouse normal cells. Sindbis virus is known to infect mammalian cells using the Mr 67,000 laminin receptor. Immunohistochemical staining of tumor cells indicates that laminin receptor is elevated in tumor versus normal cells. Down-regulated expression of laminin receptor with small interfering RNA significantly reduces the infectivity of Sindbis vectors. Tumor overexpression of the laminin receptor may explain the specificity and efficacy that Sindbis vectors demonstrate for tumor cells in vivo. We show that incorporation of antitumor cytokine genes such as interleukin-12 and interleukin-15 genes enhances the efficacy of the vector. These results suggest that Sindbis viral vectors may be promising agents for both specific detection and growth suppression of metastatic ovarian cancer.

Laminin-induced signaling in tumor cells: the role of the M(r) 67,000 laminin receptor.

The expression of the M(r) 67,000 laminin receptor, a nonintegrin laminin receptor, was found to be up-regulated in neoplastic cells and to directly correlate with invasion and metastatic potential. In the present study, we investigated the role of laminin receptor in mediating laminin effects and the involvement of the mitogen-activated protein kinases (MAPK) cascades and dual-specificity phosphatases in laminin signaling in human melanoma cells. Using stable transfection of A375SM melanoma cells, we established lines expressing reduced or elevated laminin receptor. The antisense-transfected cells demonstrated reduced attachment to laminin and reduced invasion through Matrigel-coated filters. In addition, both matrix metalloproteinase-2 (MMP-2) mRNA expression and activity were significantly reduced in the antisense-transfected cells. Antisense-transfected cells showed a reduction in mRNA level of the alpha6B integrin subunit isoform, whereas no change in the mRNA level of the alpha6A isoform was observed. We found that exogenous laminin reduced the phosphorylated (active) form of extracellular signal-regulated kinase, c-Jun NH(2)-terminal protein kinase, and p38 in all of the cells, irrespective of the expression of the laminin receptor. Furthermore, the phosphorylation of extracellular signal-regulated kinase, c-Jun NH(2)-terminal protein kinase, and p38 was significantly higher in the cell lines expressing reduced laminin receptor, regardless of the exposure to exogenous laminin. This increase of MAPK phosphorylation was accompanied by a significant reduction in MKP-1 phosphatase mRNA level and a significant increase in PAC-1 phosphatase mRNA level. In conclusion, our results confirm the involvement of the laminin receptor in different mechanisms related to tumor dissemination and provide first evidence of the involvement of MAPK and dual-specificity phosphatases in its signal transduction pathway.

Expression of laminin receptors in schwann cell differentiation: evidence for distinct roles.

Schwann cells require laminin-2 throughout nerve development, because mutations in the alpha2 chain in dystrophic mice interfere with sorting of axons before birth and formation of myelin internodes after birth. Mature Schwann cells express several laminin receptors, but their expression and roles in development are poorly understood. Therefore, we correlated the onset of myelination in nerve and synchronized myelinating cultures to the appearance of integrins and dystroglycan in Schwann cells. Only alpha6beta1 integrin is expressed before birth, whereas dystroglycan and alpha6beta4 integrin appear perinatally, just before myelination. Although dystroglycan is immediately polarized to the outer surface of Schwann cells, alpha6beta4 appears polarized only after myelination. We showed previously that Schwann cells lacking beta1 integrin do not relate properly to axons before birth. Here we show that the absence of beta1 before birth is not compensated by other laminin receptors, whereas coexpression of both dystroglycan and beta4 integrin is likely required for beta1-null Schwann cells to myelinate after birth. Finally, both beta1-null and dystrophic nerves contain bundles of unsorted axons, but they are predominant in different regions: in spinal roots in dystrophic mice and in nerves in beta1-null mice. We show that differential compensation by laminin-1, but not laminin receptors may partially explain this. These data suggest that the action of laminin is mediated by beta1 integrins during axonal sorting and by dystroglycan, alpha6beta1, and alpha6beta4 integrins during myelination.

Expression of the high-affinity laminin receptor (67 kDa) in normal human skin and appendages.

The interaction of cells with extracellular matrix components plays a significant role in the regulation of cell biology. Laminin is a large glycoprotein involved in fundamental interactions between cells and the basement membrane. Several cell surface receptors are responsible for cell-matrix interactions. The 67 kDa high affinity laminin receptor, 67LR, is involved in the adhesion of normal cells to the laminin network and is also associated with the metastatic phenotype of some tumoral cells. We have investigated the expression of laminin and of the 67LR in normal human skin using immunoperoxidase staining. Twenty samples of skin were analyzed. Antibody against laminin reacted in a continuous linear band at the dermal-epidermal junction, as well as basement membranes of hair follicles, sebaceous and eccrine sweat glands, and dermal blood vessels. The epidermis and the follicular epithelium were negative for laminin. The 67LR seemed not to be expressed on the basal surface of basal keratinocytes. The major expression of this receptor may be detected in the upper half of the spinous layer and in the granular layer. The cells of the outer root sheath in hair follicle showed the same immunohistochemical pattern described for epidermis. In sebaceous glands and in eccrine sweat glands the secreting epithelium was positive. Endothelial cells of dermal blood vessels were routinely positive for 67LR. We observed that the expression of the 67LR in normal human skin is mostly located in epidermal areas in which the keratinizing process was particularly advanced.

Expression of the Mr 67,000 laminin receptor is an adverse prognostic indicator in human thyroid cancer: an immunohistochemical study.

Increased expression of the Mr 67,000 laminin receptor (LR) is a consistent event which appears as cancer cells acquire an invasive and metastatic phenotype. The Mr 67,000 LR is one of the many laminin-binding proteins able to interact with the major glycoprotein of basement membranes, laminin. The recent development of a specific monoclonal antibody directed against the Mr 67,000 LR MLuC5 has allowed us to study large retrospective groups of human cancers with the aim of correlating the Mr 67,000 LR expression to the clinical, pathological, and survival data of the patients. A significant correlation has already been established between the increased expression of Mr 67,000 LR and survival of patients with breast, colon, ovary, lung, and endometrial cancers. In this study, we investigated the possibility that the detection of Mr 67,000 LR in thyroid human cancers could also be of prognostic value. We analyzed the expression of Mr 67,000 LR with immunohistochemistry using MLuC5 antibodies in paraffin sections of 40 benign and 170 malignant thyroid human tumors. We found that Mr 67,000 LR was not usually detectable in normal thyroid tissues adjacent to the lesion. Only 3 of the 40 thyroid adenomas examined (7.5%) presented cells positive for Mr 67,000 LR. For the malignant thyroid tumors examined, we found that 22.3% of papillary thyroid carcinomas, 38% of follicular thyroid carcinomas, 40% of poorly differentiated carcinomas, 25% of medullary carcinomas, and 58.3% of anaplastic carcinomas expressed a high level of Mr 67,000 LR. Although no correlation between the Mr 67,000 LR expression and survival was found in patients with follicular thyroid carcinomas, papillary thyroid carcinomas, anaplastic carcinomas, and medullary carcinomas, there was a significant correlation in primary thyroid cancers. Our data represent the first extensive study of the Mr 67,000 LR expression in human thyroid cancers and strongly suggest that its detection could be of prognostic value in the investigation of primary thyroid cancers.

Altered expression of metastasis-associated and regulatory molecules in effusions from breast cancer patients: a novel model for tumor progression.

PURPOSE: The aim of this study was to characterize phenotypic alterations along the progression of breast carcinoma from primary tumor to pleural effusion through analysis of the expression of proteases, laminin receptors (LRs), and transcription factors involved in invasion and metastasis. EXPERIMENTAL DESIGN: The material studied consisted of 60 malignant pleural effusions from breast cancer patients and 68 corresponding solid tumors (37 primary and 31 metastatic tumors). Expression of matrix metalloproteinases [MMPs (MMP-1, MMP-2, MMP-9, and MMP-14)], the MMP inhibitor tissue inhibitor of metalloproteinase-2, the MMP inducer EMMPRIN, the 67-kDa LR, the alpha6 integrin subunit, and the transcription factors AP-2, Ets-1, and PEA3 was studied using immunohistochemistry, mRNA in situ hybridization, reverse transcription-polymerase chain reaction, zymography, and flow cytometry. Hormone receptor (estrogen receptor and progesterone receptor) status and c-erbB-2 status were also studied. RESULTS: Significantly reduced estrogen receptor (P < 0.001) and progesterone receptor (P = 0.001) expression was seen in effusions compared with primary tumors, with opposite findings for c-erbB-2 (P = 0.003). Tumor cell MMP-2 protein expression in effusions was higher than that in primary tumors (P < 0.001) and lymph node metastases (P = 0.01). In situ hybridization demonstrated higher MMP-2 (P = 0.007), PEA3 (P = 0.038), and EMMPRIN (P = 0.026) mRNA expression in effusions. The time to progression from primary tumor to effusion was significantly shorter for patients whose primary tumors expressed MMP-1 (P = 0.016) and who expressed the 67-kDa LR protein in primary tumor (P = 0.007) and effusion (P = 0.015). CONCLUSIONS: Our data provide documented evidence of molecular events that occur during the progression of breast carcinoma from primary tumor to effusion. The coordinated up-regulation of MMP-2 and Ets transcription factors in carcinoma cells in effusions is in full agreement with our previous reports linking these factors to poor prognosis in ovarian cancer. The rapid progression to effusion in cases showing MMP-1 and 67-kDa LR expression in primary tumor cells links aggressive clinical behavior with expression of metastasis-associated molecules in this setting.