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Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a pan-negative regulator of receptor tyrosine kinase (RTK) signaling and a tumor suppressor in several cancers, but its involvement in melanoma is largely unexplored. Here, we aim to determine the role of LRIG1 in melanoma tumorigenesis, RTK signaling, and BRAF inhibitor resistance. We find that LRIG1 is downregulated during early tumorigenesis and that LRIG1 affects activation of the epidermal growth factor receptor (EGFR) in melanoma cells. LRIG1-dependent regulation of EGFR signaling is evolutionary conserved to the roundworm C. elegans, where negative regulation of the EGFR-Ras-Raf pathway by sma-10/LRIGcompletely depends on presence of the receptor let-23/EGFR. In a cohort of metastatic melanoma patients, we observe an association between LRIG1 and survival in the triple wild-type subtype and in tumors with high EGFR expression. During in vitro development of BRAF inhibitor resistance, LRIG1 expression decreases; and mimics LRIG1knockout cells for increased EGFR expression. Treating resistant cells with recombinant LRIG1 suppresses AKT activation and proliferation. Together, our results show that sma-10/LRIGis a conserved regulator of RTK signaling, add to our understanding of LRIG1 in melanoma and identifies recombinant LRIG1 as a potential therapeutic against BRAF inhibitor-resistant melanoma.

Mutations in leucine-rich-repeats and immunoglobulin-like-domains 2 (LRIG2)or in heparanase 2 (HPSE2)cause urofacial syndrome, a devastating autosomal recessive disease of functional bladder outlet obstruction. It has been speculated that urofacial syndrome has a neural basis, but it is unknown whether defects in urinary bladder innervation are present. We hypothesized that urofacial syndrome features a peripheral neuropathy of the bladder. Mice with homozygous targeted Lrig2mutations had urinary defects resembling those found in urofacial syndrome. There was no anatomical blockage of the outflow tract, consistent with a functional bladder outlet obstruction. Transcriptome analysis revealed differential expression of 12 known transcripts in addition to Lrig2, including 8 with established roles in neurobiology. Mice with homozygous mutations in either Lrig2or Hpse2had increased nerve density within the body of the urinary bladder and decreased nerve density around the urinary outflow tract. In a sample of 155 children with chronic kidney disease and urinary symptoms, we discovered novel homozygous missense LRIG2variants that were predicted to be pathogenic in 2 individuals with non-syndromic bladder outlet obstruction. These observations provide evidence that a peripheral neuropathy is central to the pathobiology of functional bladder outlet obstruction in urofacial syndrome, and emphasize the importance of LRIG2and heparanase 2for nerve patterning in the urinary tract.

The present study was conducted to investigate if chromosome band 3p14 is of any pathogenic significance in the malignant process of breast cancer. Genetic studies have implicated a tumour suppressor gene on chromosome arm 3p and we have proposed LRIG1at 3p14 as a candidate tumour suppressor. The LRIG1gene encodes an integral membrane protein that counteracts signalling by receptor tyrosine kinases belonging to the ERBB family. LRIG1 mRNA and protein are expressed in many tissues, including breast tissue. In the present report we analysed the LRIG1gene by fluorescence in situhybridisation (FISH), LRIG1mRNA by quantitative RT-PCR, and LRIG1 protein by western blot analysis. Two tumour series were analysed; one series consisted of 19 tumour samples collected between 1987 and 1995 and the other series consisted of 9 tumour samples with corresponding non-neoplastic breast tissues collected consecutively. The LRIG1gene showed increased copy number in 11 out of 28 tumours (39%) and only one tumour showed a deletion at this locus. Increased LRIG1copy number was associated with increased levels of LRIG1mRNA (two of three tumours) and protein (four of four tumours) in the tumours compared to matched non-neoplastic breast tissue, as assessed by RT-PCR and western blot analysis. The molecular function of LRIG1 as a negative regulator of ERBB receptors questions the biological significance of increased LRIG1copy number in breast cancer. We propose that a common, but hitherto unrecognised, breast cancer linked gene is located within an amplicon containing the LRIG1locus at 3p14.3.

Introduction The present study was conducted to investigate if chromosome band 3p14 is of any pathogenic significance in the malignant process of breast cancer. Genetic studies have implicated a tumour suppressor gene on chromosome arm 3p and we have proposed LRIG1 at 3p14 as a candidate tumour suppressor. The LRIG1 gene encodes an integral membrane protein that counteracts signalling by receptor tyrosine kinases belonging to the ERBB family. LRIG1 mRNA and protein are expressed in many tissues, including breast tissue.Methods In the present report we analysed the LRIG1 gene by fluorescence in situ hybridisation (FISH), LRIG1 mRNA by quantitative RT-PCR, and LRIG1 protein by western blot analysis. Two tumour series were analysed; one series consisted of 19 tumour samples collected between 1987 and 1995 and the other series consisted of 9 tumour samples with corresponding non-neoplastic breast tissues collected consecutively.Results The LRIG1 gene showed increased copy number in 11 out of 28 tumours (39%) and only one tumour showed a deletion at this locus. Increased LRIG1 copy number was associated with increased levels of LRIG1 mRNA (two of three tumours) and protein (four of four tumours) in the tumours compared to matched non-neoplastic breast tissue, as assessed by RT-PCR and western blot analysis.Conclusion The molecular function of LRIG1 as a negative regulator of ERBB receptors questions the biological significance of increased LRIG1 copy number in breast cancer. We propose that a common, but hitherto unrecognised, breast cancer linked gene is located within an amplicon containing the LRIG1 locus at 3p14.3.

The detailed mechanisms behind the resistance of malignant gliomas to therapy are not known. Inherent resistance to apoptosis is, however, one plausible explanation. In the present study we tried to delineate the molecular defects and to induce apoptosis by inducible caspases in three apparently apoptosis resistant glioma cell lines. U-105 MG, U-251 MG, and SF-767 were resistant to Fas-induced apoptosis as shown by the lack of Fas-induced cell death, morphological changes, annexin-V reactivity, Parp cleavage, caspase-3 cleavage, and caspase-3 activation. The glioma cells showed no consistent down-regulation of the pro-apoptotic proteins Fas, Fadd, caspase-3, caspase-8, caspase-9, Apaf-1, Bid, Bad, or Bax, and no consistent up-regulation of the anti-apoptotic proteins Bcl-x or Bcl-2. In U-105 MG, Fas was, however, not detected at the cell surface indicating intracellular retention. To assess if the apoptotic blocks could be by-passed, we introduced the so-called artificial death switches, i.e., inducible caspases and Fadd, into the glioma cells. Synthetic activation of inducible caspase-3, but not of caspase-8, resulted in apoptosis in the three glioma cell lines and inducible Fadd induced apoptosis in SF-767. The results were consistent with a block in the apoptotic signaling pathways of glioma cells between caspase-8 and caspase-3 activation, and that inducible Fadd could induce caspase-8 independent apoptosis in some cells. Apparently resistant glioma cells could thus be induced to undergo apoptosis by activation of appropriate death switches. This might have implications for the design of future therapeutic strategies.

Irradiation is one of the cornerstones used in the treatment of malignant glioma. However, the effect is modest and glioma cells generally display a pronounced radio-resistance. In this study, the effect of irradiation, alone and in combination with the antimicrotubule drug estramustine (EaM), was investigated in vitro using the BT4C rat glioma cell line, and in vivo the BT4C rat intracerebral glioma model was used. Apoptosis was detected by analysing DNA laddering, in situ end labelling (ISEL) and Annexin V reactivity. In addition, phosphorylation status of MAPK, JNK, p38, and AKT, proteins involved in pro- and anti-apoptotic signalling pathways was analysed by Western blotting. Irradiation did not induce apoptosis, neither in vitro nor in vivo. EaM, however, induced apoptosis in vivo and in vitro, regardless of whether EaM was given alone, before or after irradiation. When BT4C cells were treated with the caspase-3 inhibitor Ac-DEVD-CHO prior to EaM, the number of apoptotic cells was decreased, indicating an involvement of caspase-3. The signalling pathways regulating apoptosis are complex and involve kinases such as MAPK, JNK, p38 and AKT. Irradiation did not induce any changes in the expression levels or phosphorylation status of these proteins. On the other hand, the phosphorylation level of AKT was reduced after EaM treatment, which might, in part, propose how EaM induces apoptosis in glioma cells.

Estramustine is a chemotherapeutic drug, used in the treatment of prostatic carcinoma. In the prostate, it binds specifically to a 46 kDa glycoprotein called estramustine-binding protein (EMBP), which consists of three polypeptide components; C1, C2, and C3, each coded for by a specific gene. Expression of EMBP and binding of estramustine has also been detected in malignant glioma in both rats and humans. Elevated levels of this protein in astrocytoma have proved to correlate with poor prognosis. In the present work, expression of all three polypeptide components of EMBP was confirmed in an orthotopic rat glioma model with nested reverse transcriptase PCR and Western blot (molecular weights of 8, 10, and 12 kDa). Specific binding of estramustine with a Kdof 40 for male and 50 for female rats, and a total number of binding sites of 0.7 and 0.4 pmol/mg proteins for male and female rats respectively, was demonstrated with Scatchard plot analysis. These binding characteristics are similar to those of prostatic EMBP. Further studies to elucidate how EMBP expression affects the effect of estramustine treatment, and its putative prognostic value is of special clinical interest. The confirmation of BMBP expression in BT4C rat glioma demonstrates its suitability as a model system for such studies.

Human immunodeficiency virus type 1 (HIV-1)-mediated syncytium formation is recognized as being highly dependent on intercellular adhesion molecule (ICAM)-1–leukocyte function-associated molecule 1 (LFA)-1 interaction, whereas the process of infection with cell-free virions is independent of such complementary interaction. Our group has recently demonstrated that an antibody-mediated induction of the high affinity state of LFA-1 for ICAM-1 renders target T cells more prone to HIV-1-dependent syncytium formation and infection by ICAM-1-bearing virions. To further substantiate these results, we made use of mutant cell lines expressing LFA-1 in either a low (parental HPB-ALL and HAmut) or a high affinity state for ICAM-1 (HAP4) and compared syncytium formation and virus infection. Cells expressing the activated form of LFA-1 were found to be more susceptible to HIV-1-induced syncytium formation and to infection by ICAM-1-bearing HIV-1 particles. The observed increase was solely due to the LFA-1 activation state because it was abrogated by anti-LFA-1 or anti-ICAM-1 antibodies and not due to variations in surface expression of LFA-1, CD4, or the chemokine coreceptor CXCR4. However, a linear relation was seen between the level of CXCR4 surface expression and susceptibility to syncytium formation/virus infection when ICAM-1–LFA-1 interaction was either absent (i.e., infection with ICAM-1-negative virions) or abrogated (treatment with anti-LFA-1 or anti-ICAM-1 antibodies). These results emphasize the important role of the LFA-1 activation state with respect to virus-induced syncytium formation and HIV-1 infection.

One of the aims of this study was to evaluate an information campaign carried out in three geographical areas of Sweden in the winter of 1992‐93. The campaign was intended to increase public awareness of organ donation and to increase the signing of donor cards. Another objective was to test the effects of different kinds of information. These were: A) an extensive “package” of information including training of key groups, lecturing at meetings and exhibitions, and advertisements of donor cards: B) a brochure to households including two donor cards; and C) a combination of A and B. Yet another aim was to reassess public opinion on transplantation issues, which had been surveyed before in 1987, 1988, and 1990. Random samples of the population in three campaign areas and a control sample were surveyed before and after the campaign, altogether 5600 persons. The average response rate was 69% (1992) and 68% (1993). In the two areas where the brochure had been distributed to the households, the rate of donor card holders had more than doubled (from 3% and 5% to 13% and 12%). In the two areas where the brochure had not been distributed, the rate was unchanged (5%). In the “brochure areas” also a somewhat larger number of people had informed their relatives about their decisions, compared with people in the other areas. In all campaign areas considerably more people were aware of the cards than in the control area. No attitude changes could be shown in any area. Thus, the mailed brochure was the most effective in increasing people's signing of the donor cards and informing their families. The more elaborate campaign could possibly have long‐term effects, but this remains to be studied. The attitudes toward organ donation have been rather constant in Sweden from 1987 to 1993, with only slight variations. A “frankness gradient” was confirmed in this study as well as in an earlier study, where those who were negative toward donation of their own organs had discussed this issue least with their families, followed by those who were undecided, whereas those who were positive (and above all those positive who were card‐holders) had discussed this the most with their relatives.