Search

Background: We aim to implement an immune cell score model in routine clinical practice for resected non-small-cell lung cancer (NSCLC) patients (NCT03299478). Molecular and genomic features associated with immune phenotypes in NSCLC have not been explored in detail., Patients and Methods: We developed a machine learning (ML)-based model to classify tumors into one of three categories: inflamed, altered, and desert, based on the spatial distribution of CD8+ T cells in two prospective (n = 453; TNM-I trial) and retrospective (n = 481) stage I-IIIA NSCLC surgical cohorts. NanoString assays and targeted gene panel sequencing were used to evaluate the association of gene expression and mutations with immune phenotypes., Results: Among the total of 934 patients, 24.4% of tumors were classified as inflamed, 51.3% as altered, and 24.3% as desert. There were significant associations between ML-derived immune phenotypes and adaptive immunity gene expression signatures. We identified a strong association of the nuclear factor-κB pathway and CD8+ T-cell exclusion through a positive enrichment in the desert phenotype. KEAP1 [odds ratio (OR) 0.27, Q = 0.02] and STK11 (OR 0.39, Q = 0.04) were significantly co-mutated in non-inflamed lung adenocarcinoma (LUAD) compared to the inflamed phenotype. In the retrospective cohort, the inflamed phenotype was an independent prognostic factor for prolonged disease-specific survival and time to recurrence (hazard ratio 0.61, P = 0.01 and 0.65, P = 0.02, respectively)., Conclusions: ML-based immune phenotyping by spatial distribution of T cells in resected NSCLC is able to identify patients at greater risk of disease recurrence after surgical resection. LUADs with concurrent KEAP1 and STK11 mutations are enriched for altered and desert immune phenotypes., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The lack of animal models for some human diseases precludes our understanding of disease mechanisms and our ability to test prospective therapies in vivo. Generation of kidney organoids from Tuberous Sclerosis Complex (TSC) patient-derived-hiPSCs allows us to recapitulate a rare kidney tumor called angiomyolipoma (AML). Organoids derived from TSC2 -/- hiPSCs but not from isogenic TSC2 +/- or TSC2 +/+ hiPSCs share a common transcriptional signature and a myomelanocytic cell phenotype with kidney AMLs, and develop epithelial cysts, replicating two major TSC-associated kidney lesions driven by genetic mechanisms that cannot be consistently recapitulated with transgenic mice. Transplantation of multiple TSC2 -/- renal organoids into the kidneys of immunodeficient rats allows us to model AML in vivo for the study of tumor mechanisms, and to test the efficacy of rapamycin-loaded nanoparticles as an approach to rapidly ablate AMLs. Collectively, our experimental approaches represent an innovative and scalable tissue-bioengineering strategy for modeling rare kidney disease in vivo., (© 2021. The Author(s).)

Rheb is a Ras family GTPase, which binds to and activates mammalian target of rapamycin complex 1 (mTORC1) when GTP loaded. Recently, cancer genome sequencing efforts have identified recurrent Rheb Tyr35Asn mutations in kidney and endometrial carcinoma. Here we show that Rheb-Y35N causes not only constitutive mTORC1 activation, but sustained activation of the MEK-ERK pathway in a TSC1/TSC2/TBC1D7 protein complex and mTORC1-independent manner, contributing to intrinsic resistance to rapamycin. Rheb-Y35N transforms NIH3T3 cells, resulting in aggressive tumor formation in xenograft nude mice, which could be suppressed by combined treatment with rapamycin and an extracellular signal-regulated kinase (ERK) inhibitor. Furthermore, Rheb-Y35N inhibits AMPKα activation in response to nutrient depletion or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), leading to attenuated phosphorylation of BRAF-S729 and retained mitogen-activated protein kinase (MAPK) activation. Finally, we demonstrate that Rheb-WT can bind AMPK to facilitate AMPK activation, whereas Rheb-Y35N competitively binds AMPK, impairing AMPK phosphorylation. In summary, our findings indicate that Rheb-Y35N is a dominantly active tumor driver that activates both mTORC1 and MAPK to promote tumor growth, suggesting a combination of mTORC1 and MAPK inhibitors may be of therapeutic value in patients whose cancers sustain this mutation.

Tuberous sclerosis complex 1 (TSC1) and TSC2 are suppressors of mechanistic target of rapamycin (mTOR). mTOR is the major component of two protein complexes: mTOR complex 1 (mTORC1) and mTORC2. Inactive mutation of either TSC1 or TSC2 unleashes mTOR signaling and consequently causes TSC, a benign tumor syndrome affecting multiple organs. We report here that expression of αB-crystallin was upregulated in Tsc1-/- or Tsc2-/- mouse embryonic fibroblasts, Eker rat uterine leiomyoma-derived Tsc2-deficient ELT3 cells, mutant Tsc2-associated mouse kidney tumors, and human lung lymphangioleiomyomatosis nodules. αB-crystallin was transcriptionally activated by mTOR complex 2 (mTORC2): nuclear factor-kappa B (NFκB) signaling cascade. The augmented αB-crystallin was critical for the migration, invasion and apoptotic resistance of Tsc2-defective cells. Disruption of αB-crystallin suppressed Tsc2-null cell proliferation and tumorigenesis. Therefore, enhanced αB-crystallin has an essential role in TSC1/2 complex deficiency-mediated tumorigenesis, and inhibition of αB-crystallin may complement the current therapy for TSC.

Mesothelioma is diagnosed in ∼2500 patients in the United States every year, most often arising in the pleural space, but also occurring as primary peritoneal mesothelioma. The vast majority of patients with mesothelioma die of their disease within 3 years. We developed a new mouse model of mesothelioma by bladder or intraperitoneal injection of adenovirus Cre into mice with conditional alleles of each of Tp53 and Tsc1. Such mice began to develop malignant ascites about 6 months after injection, which was due to peritoneal mesothelioma, on the basis of tumor morphology and immunohistochemical staining. Mesothelioma cell lines were established, which showed loss of both Tsc1 and Tp53, with mammalian target of rapamycin complex (mTORC)1 activation. Treatment of mice with malignant ascites due to mesothelioma with rapamycin led to a marked reduction in ascites, extended survival and a 95-99% reduction in the mesothelioma tumor volume, in comparison with vehicle-treated mice. To see whether TSC1/TSC2 loss was a common genetic event in human mesothelioma, we examined nine human mesothelioma cell lines, and found that four of nine showed persistent activation of mTORC1, although none had loss of TSC1 or TSC2. A tissue microarray analysis of 198 human mesothelioma specimens showed that 33% of cases had reduced TSC2 expression and 60% showed activation of mTOR, indicating that mTOR activation is common in human mesothelioma, suggesting that it is a potential therapeutic target.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, gefitinib and erlotinib are effective therapies against mutant non-small cell lung cancers (NSCLCs). Treatment is limited by the development of resistance in part explained by the gain of a secondary EGFR mutation, T790M, at the gatekeeper residue. Irreversible EGFR inhibitors, including PF00299804, are effective in vitro and in vivo against EGFR mutant tumors that contain EGFR T790M and are currently under clinical development. In this study, we generate models of resistance to PF00299804, using cell lines with EGFR T790M and show that the PF00299804-resistant models develop focal amplification of EGFR that preferentially involves the T790M-containing allele. These PF00299804-resistant cell lines remain dependent on EGFR for growth as downregulation of EGFR by shRNA compromises their viability. We show that resistance to PF00299804 arises, at least in part, through selection of a pre-existing EGFR T790M-amplified clone both in vitro and using a xenograft model in vivo. Our findings show that EGFR T790M is a common resistance mechanism to both reversible, and when amplified, the irreversible EGFR kinase inhibitors further emphasizing the need to develop more potent therapies against EGFR T790M. These findings can be used to guide studies of patient tumor specimens from ongoing clinical trials of irreversible EGFR kinase inhibitors.

Germline TSC1 or TSC2 mutations cause tuberous sclerosis complex (TSC), a hamartoma syndrome with lung involvement. To explore the potential interaction between TSC1 and KRAS activation in lung cancer, mice in which Tsc1 loss and Kras(G12D) expression occur in a small fraction of lung epithelial cells were generated. Mice with a combined Tsc1-Kras(G12D) mutation had dramatically reduced tumor latency (median survival: 11.6-15.6 weeks) in comparison with Kras(G12D) alone mutant mice (median survival: 27.5 weeks). Tsc1-Kras(G12D) tumors showed consistent activation of mTOR (mammalian target of rapamycin)C1 and responded to treatment with rapamycin, leading to significantly improved survival, whereas rapamycin had minor effects on cancers in Kras(G12D) alone mice. Loss of heterozygosity for TSC1 or TSC2 was found in 22% of 86 human lung cancer specimens. However, none of the 80 lung cancer lines studied showed evidence of the lack of expression of either TSC1 or TSC2 or a signaling pattern corresponding to complete loss. These data indicate that Tsc1 loss synergizes with the Kras mutation to enhance lung tumorigenesis in the mouse, but that this is a rare event in human lung cancer. Rapamycin may have unique benefit for patients with lung cancer, for whom the TSC1/TSC2 function is limited.

Tuberous Sclerosis Complex (TSC) is a multi-system disorder that is highly variable in its clinical presentation. Current molecular diagnostic methods permit identification of mutations in either TSC1 or TSC2 in 75-85% of TSC patients. Here we examine the clinical characteristics of those TSC patients who have no mutation identified (NMI). A retrospective review of our patient population that had comprehensive testing for mutations in TSC1/TSC2 identified 23/157 (15%) that were NMI. NMI patients had a lower incidence of brain findings on imaging studies, neurological features, and renal findings than those with TSC2 mutations. In contrast, NMI patients had a lower incidence of seizures than TSC patients with TSC1 mutations, but had a higher incidence of both renal angiomyolipomas and pulmonary lymphangioleiomyomatosis. This distinct constellation of findings suggest that NMI patients may have a unique molecular pathogenesis, different from that seen in TSC patients with the usual mutations in TSC1 and TSC2. We suggest that the mechanisms of disease in these patients include both mosaicism for a TSC2 mutation, and unusual non-coding region mutations in TSC2.

Chronic obstructive pulmonary disease (COPD) is associated with a systemic inflammatory state, marked by elevations in serum inflammatory markers including C-reactive protein (CRP). The present study sought to determine epidemiological predictors of CRP levels, to estimate the genetic influence on CRP levels, and to identify genetic variants that affect CRP in a family-based study of COPD. CRP was measured by a high-sensitivity assay in participants from the Boston Early-Onset COPD Study. Predictors of CRP level were determined using multilevel linear models. Variance component analysis was used to estimate heritability and to perform genome-wide linkage analysis for CRP levels. Two variants in the surfactant protein B (SFTPB) gene were tested for association with CRP levels. Increased age, female sex, higher body mass index, greater smoking pack-yrs and reduced forced expiratory volume in one second were all associated with increased CRP levels. There was a significant genetic influence on CRP (heritability = 0.25). Genome-wide linkage analysis revealed several potentially interesting chromosomal regions, though no significant evidence for linkage was found. A short tandem repeat marker near SFTPB was significantly associated with CRP levels. There is a genetic influence on C-reactive protein levels in chronic obstructive pulmonary disease patients. Preliminary evidence suggests an association of the surfactant protein B gene with systemic inflammation in chronic obstructive pulmonary disease.

The aim of this research was to assess whether common genetic variants within the C-reactive protein gene (CRP) are related to the degree of acute rise in plasma C-reactive protein (CRP) levels following an acute coronary syndrome (ACS). While polymorphisms within CRP are associated with basal CRP levels in healthy men and women, less is known about the relationship of such genetic variants and the degree of CRP rise during and after acute ischemia. Plasma CRP is associated with increased rates of recurrent coronary events. We evaluated seven common genetic variants within CRP and assessed their relationship to the degree of rise in CRP levels immediately following an acute coronary syndrome in 1827 European American patients. Variants in the putative promoter region, -757T > C and -286C > T > A, were associated with the highest CRP elevations after ACS. Patients with two copies of the A allele of SNP -286C > T > A had median CRP values of 76.6 mg/L, compared to 11.1 mg/L in patients with no copies of the rare variant (p-value <0.0001), post ACS. The lowest CRP values were found for patients with minor alleles of the exonic 1059G > C and the 3'untranslated region 1846G > A SNPs. For example, patients homozygous for the minor allele of 1059G > C had 71% lower median CRP values than those homozygous for the major allele [3.5 vs 12.0 mg/L, p < 0.0001]. These trends persisted in the chronic stable phase after ischemia had resolved, and after adjustment for infarct size by peak creatinine kinase levels and clinical status by Killip class. Assessment of CRP genetic variants identified patients with higher and lower CRP elevation after acute coronary syndrome.

A genetic link between lipid metabolism and inflammation has been suggested by the association between variation in the APOE gene and plasma C-reactive protein (CRP). This association was confirmed among Caucasians and extended to an African-American population, and the well-known associations of APOE variation with LDL-C and apoE protein were also observed. While eight common variants in APOE were examined, the association with CRP involved primarily the two nonsynonymous SNPs that define the major epsilon2, epsilon3, and epsilon4 alleles. In particular, the strongest link involved lower CRP levels among carriers of the APOE epsilon4 allele that also contributes to the risk of cardiovascular and Alzheimer's diseases as well as to higher lipid levels. A lesser effect was characterized by lower CRP levels among carriers of a subtype of the epsilon3 allele. The magnitude of the association with plasma CRP was at least as great as the effect of variation in the CRP gene itself. Quantitative analysis suggested that the effect on CRP is more likely a consequence of intrinsic functional differences among the E2, E3, and E4 apoE proteins than different levels of apoE protein or LDL-C in the plasma.

C-reactive protein (CRP) is a well-documented marker of atherosclerotic cardiovascular disease risk. We resequenced CRP to identify a comprehensive set of common SNP variants, then studied and replicated their association with baseline CRP level among apparently healthy subjects in the Women's Health Study (WHS; n = 717), Pravastatin Inflammation/CRP Evaluation trial (PRINCE; n = 1,110) and Physicians' Health Study (PHS; n = 509) cohorts. The minor alleles of four SNPs were consistently associated in all three cohorts with higher CRP, while the minor alleles of two SNPs were associated with lower CRP (p < 0.05 for each). Single marker and haplotype analysis in all three cohorts were consistent with functional roles for the 5'-flanking triallelic SNP -286C>T>A and the 3'-UTR SNP 1846G>A. None of the SNPs associated with higher CRP were associated with risk of incident myocardial infarction (MI) or ischemic stroke in a prospective, nested case-control study design from the PHS cohort (610 case-control pairs). One SNP, -717A>G, was unrelated to CRP levels but associated with decreased risk of MI (p = 0.001). Taken together, these data imply significant interactions between both genetic and environmental contributions to the increased CRP levels that predict a greater risk of future atherothrombotic events in epidemiological studies.

Toll-like receptor 6 (TLR6) is one of a series of highly conserved innate immune receptors. We resequenced TLR6 in DNA samples from 24 African Americans, 23 European Americans, and 24 Hispanic Americans, identifying 53 SNPs, 22 with an allele frequency >5%. Significant differences in SNP frequencies among the three populations were noted. In all, 11 SNPs caused amino-acid changes, including one with a frequency >5% in all three populations. Utilizing this SNP (Ser249Pro), we performed exploratory nested case-control disease-association studies, including one involving 56 African Americans with asthma and 93 African American controls. The minor allele of this SNP was associated with decreased risk for asthma (odds ratio 0.38, 95% CI 0.16-0.87, P=0.01), an effect consistent with the known biology of the toll-like receptors. Although replication of this finding in other, larger samples is needed, variation in TLR6 may have relevance to the pathogenesis of immunologically mediated diseases.

The authors studied nine members of a family that demonstrated a limited form of tuberous sclerosis complex (TSC). Cutaneous findings were limited to hypopigmented macules in four patients. Five family members had recurrent seizures, and three of these had migrational defects of the cerebral mantle. Mutational analysis of TSC2 indicated the presence of the novel missense change 3106T-->C, 1036S-->P in all family members with seizures. The findings suggest that this mild variant form of TSC is due to a novel TSC2 mutation.

Tuberous sclerosis (TSC) is an autosomal dominant hamartoma syndrome whose causative genes (TSC1 and TSC2) were identified 5 and 9 years ago respectively. Their encoded proteins are large, and apart from a strong binding interaction with each other, relatively little was known about their biochemical function. Recent studies in Drosophila have pinpointed a critical function for the DrosophilaTSC1/TSC2 homologues in the regulation of cell size. Epistasis experiments and a variety of biochemical studies that followed have indicated a critical function for these proteins in the highly conserved PI-3-kinase-Akt-mTOR signalling pathway.

Tuberous sclerosis complex (TSC), an autosomal dominant disease caused by mutations in either TSC1 or TSC2, is characterized by the development of hamartomas in a variety of organs. Concordant with the tumor-suppressor model, loss of heterozygosity (LOH) is known to occur in these hamartomas at loci of both TSC1 and TSC2. LOH has been documented in renal angiomyolipomas (AMLs), but loss of the wild-type allele in cortical tubers appears to be very uncommon. Analysis of second, somatic events in tumors for which the status of both TSC1 and TSC2 is known is essential for exploration of the pathogenesis of TSC-lesion development. We analyzed 24 hamartomas from 10 patients for second-hit mutations, by several methods, including LOH, scanning of all exons of both TSC1 and TSC2, promoter methylation of TSC2, and clonality analysis. Our results document loss of the wild-type allele in six of seven AMLs, without evidence of the inactivation of the second allele in many of the other lesions, including tumors that appear to be clonally derived. Laser-capture microdissection further demonstrated loss of the second allele in all three cellular components of an AML. This study thus provides evidence that, in both TSC1 and TSC2, somatic mutations resulting in the loss of wild-type alleles may not be necessary in some tumor types-and that other mechanisms may contribute to tumorigenesis in this setting.

Capping the barbed ends of actin filaments is a critical step for regulating actin-based motility in nonmuscle cells. The in vivo function of CapG, a calcium-sensitive barbed end capping protein and member of the gelsolin/villin family, has been assessed using a null Capg allele engineered into mice. Both CapG-null mice and CapG/gelsolin double-null mice appear normal and have no gross functional abnormalities. However, the loss of CapG in bone marrow macrophages profoundly inhibits macrophage colony stimulating factor-stimulated ruffling; reintroduction of CapG protein by microinjection fully restores this function. CapG-null macrophages also demonstrate approximately 50% impairment of immunoglobulin G, and complement-opsonized phagocytosis and lanthanum-induced vesicle rocketing. These motile functions are not impaired in gelsolin-null macrophages and no additive effects are observed in CapG/gelsolin double-null macrophages, establishing that CapG function is distinct from, and does not overlap with, gelsolin in macrophages. Our observations indicate that CapG is required for receptor-mediated ruffling, and that it is a major functional component of macrophage phagocytosis. These primary effects on macrophage motile function suggest that CapG may be a useful target for the regulation of macrophage-mediated inflammatory responses.

Lymphangioleiomyomatosis (LAM) and multifocal micronodular pneumocyte hyperplasia (MMPH) produce cystic and nodular disease, respectively, in the lungs of patients with tuberous sclerosis. The objective of this study was to prospectively characterize the prevalence, clinical presentation, and genetic basis of lung disease in TSC. We performed genotyping and computerized tomographic (CT) scanning of the chest on 23 asymptomatic women with tuberous sclerosis complex (TSC). Cystic pulmonary parenchymal changes consistent with LAM were found in nine patients (39%). These patients tended to be older than cyst-negative patients (31.9 +/- 7.6 yr versus 24.8 +/- 11.6 yr, p = 0.09). There was no correlation between presence of cysts and tobacco use, age at menarche, history of pregnancy, or estrogen-containing medications. Three of the cyst-positive patients had a prior history of pneumothorax. Pulmonary function studies revealed evidence of gas trapping but normal spirometric indices in the cyst-positive group. All nine cyst-positive patients had angiomyolipomas (AML), which were larger (p < 0.05) and more frequently required intervention (p = 0.08) than cyst-negative patients (8 of 14 with AMLs, p < 0.05). Ten patients (43%) had pulmonary parenchymal nodules. Pulmonary nodules were more common in women with cysts (78% versus 21%, p < 0.05), and 52% of all patients had either cystic or nodular changes. TSC2 mutations were identified in all cyst-positive patients who were tested (n = 8), whereas both TSC1 and TSC2 mutations were found in patients with nodular disease. Correlation of the mutational and radiographic data revealed one pair of sisters who were discordant for cystic disease, two mother- daughter pairs who were discordant for nodular disease, and no clear association between cyst development and a specific mutational type. This prospective analysis demonstrates that cystic and nodular pulmonary changes consistent with LAM and MMPH are common in women with TSC.

Purpose: erbB-2 and epidermal growth factor receptor (EGFR) may mediate motility via signaling that enables changes in the actin cytoskeleton. A physical basis for this motility may depend on the coexpression of gelsolin, a M(r) 80,000 actin-binding protein., Experimental Design: The expression of erbB-2, EGFR, and gelsolin was analyzed in 790 archival invasive breast cancers. These data were compared with histological, clinical, and outcome data (median follow-up, 16.3 years)., Results: Protein overexpression was observed in overlapping subsets of breast cancers (38% of cases were erbB-2+; 15% of cases were EGFR+; and 56% of cases were gelsolin+). Tumor gelsolin was associated with overexpression of erbB-2 and EGFR, as well as with an aggressive tumor phenotype. By univariate and multivariate analyses, tumor gelsolin alone was not a prognostic factor. Overexpression of all three factors significantly predicted poor clinical outcome by univariate and multivariate analyses. For example, in node-positive patients, coexpression of all three markers was associated with a 3-year disease-specific survival (as compared with erbB-2+, EGFR+, gelsolin- patients, who had a median survival of 6 years)., Conclusions: These data suggest that gelsolin coexpression may be an important additional prognostic factor in erbB-2+, EGFR+ breast cancer patients. We hypothesize that this is due to the role of gelsolin in mediating motility and invasion.

Profilins are thought to play a central role in the regulation of de novo actin assembly by preventing spontaneous actin polymerization through the binding of actin monomers, and the adding of monomeric actin to the barbed actin-filament ends. Other cellular functions of profilin in membrane trafficking and lipid based signaling are also likely. Binding of profilins to signaling molecules such as Arp2/3 complex, Mena, VASP, N-WASP, dynamin I, and others, further implicates profilin and actin as regulators of diverse motile activities. In mouse, two profilins are expressed from two distinct genes. Profilin I is expressed at high levels in all tissues and throughout development, whereas profilin II is expressed in neuronal cells. To examine the function of profilin I in vivo, we generated a null profilin I (pfn1(ko)) allele in mice. Homozygous pfn1(ko/ko) mice are not viable. Pfn1(ko/ko) embryos died as early as the two-cell stage, and no pfn1(ko/ko) blastocysts were detectable. Adult pfn1(ko/wt) mice show a 50% reduction in profilin I expression with no apparent impairment of cell function. However, pfn1(ko/wt) embryos have reduced survival during embryogenesis compared with wild type. Although weakly expressed in early embryos, profilin II cannot compensate for lack of profilin I. Our results indicate that mouse profilin I is an essential protein that has dosage-dependent effects on cell division and survival during embryogenesis.

Sensitive and automated methods for the detection of DNA sequence variation are required for a wide variety of genetic studies. Diagnostic testing in human genetic disorders is one application of such methods. Tuberous sclerosis complex (TSC) is an autosomal dominant familial tumor syndrome characterized by the development of benign tumors (hamartomas) in multiple organs (OMIM # 19110, #191092). There is a high frequency of sporadic cases and significant demand from patients and families for genetic testing information. Two TSC genes have been identified (TSC1 and TSC2) and together account for all cases [1,2]. Here we report our methods for DHPLC analysis of the TSC1 gene and demonstrate the high sensitivity of this method in a blinded analysis of 21 TSC patients with known TSC1 mutations. In this series, DHPLC detected 27/28 (96%) known TSC1 sequence variations. The only sequence variation not identified by DHPLC in this study is a mosaic case.

Tuberous sclerosis (TSC) is a relatively common hamartoma syndrome caused by mutations in either of two genes, TSC1 and TSC2. Here we report comprehensive mutation analysis in 224 index patients with TSC and correlate mutation findings with clinical features. Denaturing high-performance liquid chromatography, long-range polymerase chain reaction (PCR), and quantitative PCR were used for mutation detection. Mutations were identified in 186 (83%) of 224 of cases, comprising 138 small TSC2 mutations, 20 large TSC2 mutations, and 28 small TSC1 mutations. A standardized clinical assessment instrument covering 16 TSC manifestations was used. Sporadic patients with TSC1 mutations had, on average, milder disease in comparison with patients with TSC2 mutations, despite being of similar age. They had a lower frequency of seizures and moderate-to-severe mental retardation, fewer subependymal nodules and cortical tubers, less-severe kidney involvement, no retinal hamartomas, and less-severe facial angiofibroma. Patients in whom no mutation was found also had disease that was milder, on average, than that in patients with TSC2 mutations and was somewhat distinct from patients with TSC1 mutations. Although there was overlap in the spectrum of many clinical features of patients with TSC1 versus TSC2 mutations, some features (grade 2-4 kidney cysts or angiomyolipomas, forehead plaques, retinal hamartomas, and liver angiomyolipomas) were very rare or not seen at all in TSC1 patients. Thus both germline and somatic mutations appear to be less common in TSC1 than in TSC2. The reduced severity of disease in patients without defined mutations suggests that many of these patients are mosaic for a TSC2 mutation and/or have TSC because of mutations in an as-yet-unidentified locus with a relatively mild clinical phenotype.

Objectives: The aim of our study was to retrospectively compare the patient characteristics, the frequency and pattern of recurrent disease, and survival in patients with stage I bronchioloalveolar carcinoma and adenocarcinoma of the lung., Methods: Patients with stage I bronchioloalveolar carcinoma or adenocarcinoma other than bronchioloalveolar carcinoma resected between 1984 and 1992 with adequate clinical follow-up were studied. The clinical characteristics of the patients, extent of initial surgical resection, sites of recurrent disease, and overall survival were examined and compared between the 2 groups. The median follow-up for patients with bronchioloalveolar carcinoma and adenocarcinoma was 6.2 years and 5.9 years, respectively., Results: A total of 138 patients were identified. Thirty-three patients had bronchioloalveolar carcinoma and 105 patients had adenocarcinoma. Eleven (33%) of the patients with bronchioloalveolar carcinoma had never smoked cigarettes versus 9 (9%) of the patients with adenocarcinoma (P =.0036). There were no significant differences between patients with bronchioloalveolar carcinoma and adenocarcinoma in sex distribution and overall recurrence rate. Of the 12 patients with recurrent bronchioloalveolar carcinoma, 1 patient (8%) had extrathoracic disease develop at the site of first recurrence compared with 49% of patients with recurrent adenocarcinoma (P <.001). The 5-year survival in patients with bronchioloalveolar carcinoma and in those with adenocarcinoma was 83% and 63%, respectively (P =.04)., Conclusions: Stage I bronchioloalveolar carcinoma is more likely to occur in nonsmokers. Survival is longer in patients with bronchioloalveolar carcinoma. Further research is warranted to define the etiology, clinical course, and molecular abnormalities in patients with bronchioloalveolar carcinoma to generate more effective therapeutic approaches.

Gelsolin is an actin-binding/severing protein expressed in intracellular and secreted forms. It is a major regulator of the form and function of the actin cytoskeleton in most all cells. Here we demonstrate that female mice with a targeted deletion of the gelsolin gene (Gsn-/-) have defects in mammary gland morphogenesis. Two distinct defects were identified in the gelsolin-null mammary gland. First, the mammary anlage from Gsn-/- mice failed to elongate at the onset of puberty and remained rudimentary until approximately 9 weeks of age, early block (Gsn-/-(EB)). Second, after the mammary epithelium had filled the mammary fat pad, a complete lack of terminal branching, or late block, was observed (Gsn-/-(LB)). The Gsn-/-(EB) was seen in 70% of Gsn-/- mice and appeared to be dependent on a modifier gene(s) in addition to the loss of gelsolin. Gsn-/-(LB) was observed in all Gsn-/- mice. Terminal end buds (TEBs) were not evident in the mammary anlage from Gsn-/-(EB) mice until approximately 9 weeks of age. Cellular proliferation in the terminal ductal regions of Gsn-/-(EB) females was detected by bromodeoxyuridine incorporation, but was less than that found in the TEBs of age-matched controls. In mice deficient for gelsolin, mammary gland architecture was unaltered at the histological level. Lobuloalveolar development was delayed in response to pregnancy in mammary glands of Gsn-/- mice but was otherwise normal. Lactation and involution in the gelsolin-null animals were similar to those of wild-type mice. Transplantation of epithelium devoid of gelsolin into a wild-type (GsnWT) mammary fat pad resulted in proper arborization of the ductal tree. Transplantation of GsnWT epithelium into the Gsn-/- fat pad recapitulated the lack of terminal branching seen in Gsn-/- females. These results indicate that gelsolin is required in the mammary stroma for proper ductal morphogenesis. Our results provide the first evidence of an actin regulatory protein affecting mammary ductal growth through stromal-epithelial communication., (Copyright 2000 Academic Press.)

Over the past decade, there has been considerable progress in understanding the molecular genetics of tuberous sclerosis, a disorder characterised by hamartomatous growths in numerous organs. We review this progress, from cloning and characterising TSC1 and TSC2, the genes responsible for the disorder, through to gaining insights into the functions of their protein products hamartin and tuberin, and the identification and engineering of animal models. We also present the first comprehensive compilation and analysis of all reported TSC1 and TSC2 mutations, consider their diagnostic implications and review genotype/phenotype relationships.

Background: Cdc42 and other Rho GTPases are conserved from yeast to humans and are thought to regulate multiple cellular functions by inducing coordinated changes in actin reorganization and by activating signaling pathways leading to specific gene expression. Direct evidence implicating upstream signals and components that regulate Cdc42 activity or for required roles of Cdc42 in activation of downstream protein kinase signaling cascades is minimal, however. Also, whereas genetic analyses have shown that Cdc42 is essential for cell viability in yeast, its potential roles in the growth and development of mammalian cells have not been directly assessed., Results: To elucidate potential functions of Cdc42 mammalian cells, we used gene-targeted mutation to inactivate Cdc42 in mouse embryonic stem (ES) cells and in the mouse germline. Surprisingly, Cdc42-deficient ES cells exhibited normal proliferation and phosphorylation of mitogen- and stress-activated protein kinases. Yet Cdc42 deficiency caused very early embryonic lethality in mice and led to aberrant actin cytoskeletal organization in ES cells. Moreover, extracts from Cdc42-deficient cells failed to support phosphatidylinositol 4,5-bisphosphate (PIP(2))-induced actin polymerization., Conclusions: Our studies clearly demonstrate that Cdc42 mediates PIP(2)-induced actin assembly, and document a critical and unique role for Cdc42 in this process. Moreover, we conclude that, unexpectedly, Cdc42 is not necessary for viability or proliferation of mammalian early embryonic cells. Cdc42 is, however, absolutely required for early mammalian development.

The actin regulatory protein gelsolin cleaves actin filaments in a calcium- and polyphosphoinositide-dependent manner. Gelsolin has recently been described as a novel substrate of the cysteinyl protease caspase-3, an effector protease activated during apoptosis. Cleavage by caspase-3 generates an amino-terminal fragment of gelsolin that can sever actin filaments independently of calcium regulation. The disruption of the actin cytoskeleton by cleaved gelsolin is hypothesized to mediate many of the downstream morphological changes associated with apoptosis. In contrast, overexpression of full-length gelsolin has also been reported to inhibit apoptotic cell death upstream of the activation of caspase-3, suggesting that gelsolin may also act prior to commitment to cell death. The authors previously observed that actin stabilization by the cell permeant agent jasplakinolide enhanced cell death upon interleukin (IL)-2 or IL-3 withdrawal from growth-factor-dependent lymphocyte cell lines, and hypothesized that actin polymerization could alter the activity of gelsolin, thus enhancing apoptosis. Here the authors show that constitutive overexpression of gelsolin did not, however, inhibit or dramatically enhance apoptotic cell death upon growth-factor withdrawal, nor did it modify sensitivity to jasplakinolide. In contrast to previous reports, overexpression of gelsolin in Jurkat T cells did not prevent or delay apoptosis induced by Fas ligation or ceramide treatment. Overexpressed gelsolin protein was cleaved during apoptosis, as seen previously in this and other cell types. In these model systems, therefore, the level of gelsolin expression was not a rate-limiting determinant in commitment to or time to the morphological changes of apoptosis.

The gelsolin family of actin filament binding proteins have highly homologous structures. Gelsolin and adseverin, also known as scinderin, are the most similar members of this family, with adseverin lacking a C-terminal helix found in gelsolin. This helix has been postulated to serve as a calcium-sensitive latch, keeping gelsolin inactive. To test this hypothesis, we have analyzed the kinetics of severing by gelsolin, adseverin, and a gelsolin truncate which lacks the C-terminal latch. We find that the relationship between severing rate and calcium ion concentration differs between gelsolin and adseverin, and suggest that calcium controls one rate-limiting step in the activation of adseverin and two in the activation of gelsolin. In contrast, both proteins are activated equally by protons, and have identical severing kinetics at pHs below 6.3. The temperature sensitivity of severing by adseverin and gelsolin is remarkably different, with gelsolin increasing its severing rate 8-fold per 10 degrees C increase in temperature and adseverin increasing its rate only 2-fold per 10 degrees C increase in temperature. Analysis of the gelsolin construct lacking the C-terminal helix demonstrates that this helix is responsible for the regulatory differences between gelsolin and adseverin. These results support the C-terminal latch hypothesis for the calcium ion activation of gelsolin.

The actin cytoskeleton is an important contributor to the modulation of the cell function. However, little is known about the regulatory role of this supermolecular structure in the membrane events that take place in the heart. In this report, the regulation of cardiac myocyte function by actin filament organization was investigated in neonatal mouse cardiac myocytes (NMCM) from both wild-type mice and mice genetically devoid of the actin filament severing protein gelsolin (Gsn-/-). Cardiac L-type calcium channel currents (I(Ca)) were assessed using the whole cell voltage-clamp technique. Addition of the actin filament stabilizer phalloidin to wild-type NMCM increased I(Ca) by 227% over control conditions. The basal I(Ca) of Gsn-/- NMCM was 300% higher than wild-type controls. This increase was completely reversed by intracellular perfusion of the Gsn-/- NMCM with exogenous gelsolin. Further, cytoskeletal disruption of either Gsn-/- or phalloidin-dialyzed wild-type NMCM with cytochalasin D (CD) decreased the enhanced I(Ca) by 84% and 87%, respectively. The data indicate that actin filament stabilization by either a lack of gelsolin or intracellular dialysis with phalloidin increase I(Ca), whereas actin filament disruption with CD or dialysis of Gsn-/- NMCM with gelsolin decrease I(Ca). We conclude that cardiac L-type calcium channel regulation is tightly controlled by actin filament organization. Actin filament rearrangement mediated by gelsolin may contribute to calcium channel inactivation.

Previous studies have shown that mice lacking the actin-severing and capping protein gelsolin have defects in leukocyte and platelet function. Moreover, dermal fibroblasts from gelsolin knockout (Gsn(-)) mice showed substantially reduced motility, membrane ruffling and pinocytosis. We have generated dendritic cells (DC) from spleens of Gsn(-) mice to investigate the importance of gelsolin in antigen endocytosis and processing. We show here that Gsn(-) DC produce apparently normal membrane ruffles which can resolve to form large macropinosomes. Moreover, presentation of exogenous antigens on both MHC class II and class I molecules was equivalent in Gsn(-) and wild-type DC. Thus the major rearrangements of the actin cytoskeleton needed for DC antigen uptake and presentation can proceed in the absence of a major actin filament regulatory protein.

Tuberous sclerosis (TSC) is an autosomal dominant genetic disorder in which benign hamartomas develop in multiple organs, caused by mutations in either TSC1 or TSC2. We developed a murine model of Tsc2 disease using a gene targeting approach. Tsc2-null embryos die at embryonic days 9.5-12.5 from hepatic hypoplasia. Tsc2 heterozygotes display 100% incidence of multiple bilateral renal cystadenomas, 50% incidence of liver hemangiomas, and 32% incidence of lung adenomas by 15 months of age. Progression to renal carcinoma, fatal bleeding from the liver hemangiomas, and extremity angiosarcomas all occur at a rate of less than 10%. The renal cystadenomas develop from intercalated cells of the cortical collecting duct and uniformly express gelsolin at high levels, enabling detection of early neoplastic lesions. The tumor expression pattern of the mice is influenced by genetic background, with fewer large renal cystadenomas in the outbred Black Swiss background and more angiosarcomas in 129/SvJae chimeric mice. The slow growth of the tumors in the heterozygote mice matches the limited growth potential of the great majority of TSC hamartomas, and the influence of genetic background on phenotype correlates with the marked variability in expression of TSC seen in patients.

We evaluated denaturing high pressure liquid chromatography (DHPLC) as a scanning method for mutation detection in TSC2, and compared it to conformation-sensitive gel electrophoresis (CSGE) and single-stranded conformation polymorphism analysis (SSCP). The first 20 exons of TSC2 were amplified from 84 TSC patients and screened initially by CSGE and then by DHPLC. Optimization of DHPLC analysis of each exon was carried out by design of primers with minimum variation in the melting temperature of the amplicon, and titration of both elution gradient and temperature. CSGE analysis identified 40 shifts (21 unique) in the 84 patients and 20 exons. All of these variants were detected by DHPLC, and an additional 27 changes (14 unique) were identified. Overall 15 of 28 (54%) unique single base substitutions were detected by CSGE; all were detected by DHPLC. 25 definite or probable mutations were found in these 84 patients (30%) in exons 1-20 of TSC2. In a subsequent blinded analysis of 15 samples with 18 distinct TSC2 sequence variants originally detected by SSCP in another centre, all variants were detected by DHPLC except one where the variation occurred within the primer. Ten other (7 unique) sequence variants were detected in these samples which had not been detected by SSCP. Overall, 11 of 16 (69%) unique single base substitutions were detected by SSCP; all were detected by DHPLC. We conclude that DHPLC is superior to both CSGE and SSCP for detection of DNA sequence variation in TSC2, particularly for single base substitution mutations.

The gelsolin family of actin-modulating proteins contains seven mammalian members of which three have similar domain structure and function: gelsolin, capG, and adseverin. Previous studies have provided some information on the expression of these proteins, but no comprehensive analysis of expression during development has been performed. By in situ hybridization to murine embryo sections, we show that gelsolin expression is widespread but focal from e12.5 onward, with the exception of brain and mucosal epithelium. In contrast, CapG expression is high in mucosal epithelium, inner renal medulla, and adrenal cortex, and seen at much lower levels more broadly. Adseverin expression is even more restricted, being seen at sites of endochondral bone formation during development only, and in developing and adult outer renal medulla and intestine. In parallel analyses the three genes demonstrated patterns of expression that were complementary and non-overlapping in nearly all organs. The observations suggest new functions for these proteins in organ systems and tissues where their expression was not previously recognized. They further suggest that the proteins have distinct tissue-specific functions in modulating the actin cytoskeleton during cellular motile activities, and that such functions have diverged since the genes arose ancestrally by gene duplication. Dev Dyn 1999;215:297-307., (Copyright 1999 Wiley-Liss, Inc.)

Gelsolin, an actin-modulating protein, derived from a single gene exists in intracellular and secreted forms. A point mutation at position 187 of both forms of gelsolin causes familial amyloidosis of the Finnish type (FAF). Here, we expressed both isoforms of the wild-type and FAF mutant gelsolin in mouse embryonic gelsolin-null fibroblasts. We demonstrate that the FAF mutation does not interfere with the normal actin-modulating function of intracellular gelsolin, and that aberrant processing of secreted FAF gelsolin to FAF amyloid precursor takes place in the gelsolin-negative background. These results suggest that, in patients with FAF, symptoms are caused by the accumulation in their tissues of amyloid derived from plasma gelsolin and are not due to functional differences in cytoplasmic gelsolin.

Increased Ca2+ influx through activated N-methyl-D-aspartate (NMDA) receptors and voltage-dependent Ca2+ channels (VDCC) is a major determinant of cell injury following brain ischemia. The activity of these channels is modulated by dynamic changes in the actin cytoskeleton, which may occur, in part, through the actions of the actin filament-severing protein gelsolin. We show that gelsolin-null neurons have enhanced cell death and rapid, sustained elevation of Ca2+ levels following glucose/oxygen deprivation, as well as augmented cytosolic Ca2+ levels in nerve terminals following depolarization in vitro. Moreover, major increases in infarct size are seen in gelsolin-null mice after reversible middle cerebral artery occlusion, compared with controls. In addition, treatment with cytochalasin D, a fungal toxin that depolymerizes actin filaments, reduced the infarct size of both gelsolin-null and control mice to the same final volume. Hence, enhancement or mimicry of gelsolin activity may be neuroprotective during stroke.

Several new members of the gelsolin family have been discovered in the past year. Determination of the structure of gelsolin and identification of lysophosphatidic acid as a negative regulator provide novel functional insights. Gelsolin is an obligate downstream effector of Rac for motility in dermal fibroblasts, regulates phosphoinositide signaling pathways and ion channel function in vivo, and acts as both a regulator and effector of apoptosis.

In response to extracellular signals, cells remodel actin networks. Monomeric actin subunits at the cell's leading edge assemble into linear polymers that are cross-linked by accessory proteins into three-dimensional structures that are contracted by myosins to generate hydraulic force; elsewhere in the cell, actin networks dismantle. Actin subunit sequestering proteins prevent spontaneous actin nucleation, but not the growth of actin sub-units on to fast-growing filament ('barbed') ends, and at least half of the actin in most cells is filamentous. Therefore regulation of cellular actin assembly also requires proteins that block ('cap') actin filament barbed ends. Members of the capping protein gelsolin family also sever actin filaments mechanically. Calcium and protons activate gelsolin for severing and capping. Phosphoinositides reverse such capping, and a pathway has been defined in which receptor perturbation operates through GTP-Rac1 to stimulate the synthesis of endogenous phosphoinositides that uncap actin filaments. Other GTPases (and other signalling pathways) target phosphoinositide synthesis where other protrusions (e.g. filopodia) emerge. Cells maintain adequate, albeit compromised, locomotion in the absence of some, but not all, important machine parts. For example, gelsolin-null fibroblasts crawl using predominantly filopodia rather than lamellae. However, ABP-280 (actin-binding protein of 280 kDa), which promotes orthogonal branching of short actin filaments, seems to be necessary for membrane stability and translational locomotion. ABP-null cells hardly crawl at all, although they are viable and engage in surface movements.

Background: Tumor cell motility is an important characteristic that facilitates the multistep process of tumor metastasis. Rac, ABP-280, and gelsolin are proteins that interact with actin and are important in cell motility., Methods: The authors studied a cohort of 229 Stage I nonsmall cell lung carcinoma (NSCLC) patients who had a minimum of 3 years follow-up and had been previously analyzed for 22 clinical, pathologic, and molecular features, of which 9 had been found to provide significant prognostic information in a Cox proportional hazards model. Tumor sections were stained by the avidin-biotin complex method using monoclonal antibodies against rac, ABP-280, and gelsolin., Results: In a pilot analysis of over 50 patients each, rac and ABP-280 were found to be moderately-to-highly expressed in the majority of tumors and to provide no prognostic information. Gelsolin expression was more variable and appeared to be negatively correlated with survival in the pilot population. In the larger 229-patient population, high focal gelsolin expression was seen in 32 tumors (14%) and conferred the highest relative risk (4.04) of cancer recurrence among all factors tested, compared with tumors that had no or low gelsolin expression. Moderate focal gelsolin expression, seen in 46 patients (20%), also conferred a significant risk of cancer recurrence, with a relative risk of 2.26 compared with tumors that had no or low gelsolin expression. Consideration of average gelsolin expression and of overall survival yielded similar results., Conclusions: Gelsolin expression appears to be a significant prognostic factor for cancer recurrence in cases of Stage I NSCLC.

To understand the distinct functions of the closely related actin-severing proteins adseverin and gelsolin, we examined the expression of these proteins in detail during mouse and human development using a new highly sensitive and specific set of antibody reagents. Immunoblot analysis demonstrated that adseverin was highly expressed in mouse kidney and intestine at all stages of development and in human fetal and adult kidney. In contrast and as reported previously, gelsolin was expressed much more widely in both murine and human tissues. Immunohistochemistry on murine kidney sections revealed a predominantly differential localization of adseverin and gelsolin. Adseverin was expressed in peripolar cells, thin limbs, thick ascending limbs, and principal cells of cortical and medullary collecting ducts where it was diffusely localized in the cytoplasm. Gelsolin was expressed in the distal convoluted tubule, intercalated cells and principal cells of cortical and medullary collecting ducts, and in ureter. In the distal convoluted tubule, gelsolin showed a diffuse distribution and in principal cells of collecting ducts a localization at the basolateral pole. In intercalated cells, gelsolin localization was heterogeneous, either at the apical pole or diffusely in the cytoplasm. In human fetal and adult kidney, adseverin was expressed only in collecting ducts whereas gelsolin was expressed in thick ascending limbs and collecting ducts. In mouse and human intestine adseverin was expressed in enterocytes with a gradient of increasing expression from the duodenum to the colon, and from the crypt to the villus. The observations indicate high level expression of adseverin in specific cells of the kidney and colon, and suggest a previously unrecognized function of adseverin in epithelial cell function.

Gelsolin, an 80 kDa actin-severing protein, has been recently identified as a substrate for the cell death-promoting cysteinyl protease caspase-3 (CPP32/apopain/YAMA). We investigated the role of gelsolin and its cleavage product in apoptosis of vascular smooth muscle cells (SMC) induced by the proinflammatory cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). Treatment with a combination of IFN-gamma and TNF-alpha reduced viability of SMC in a time- and concentration-dependent manner. Immunoblotting revealed that SMC treated with the cytokines generated a 41 kDa gelsolin fragment. The gelsolin fragmentation required activation of caspase-3, as the caspase-3 inhibitor diminished cytokine-induced cell death as well as the fragmentation. Gelsolin cleavage was accompanied by a reduction in F-actin content and by a marked disruption of cell structure. Adenovirus-mediated transfection of this N-terminal gelsolin fragment into SMC altered cell morphology, reduced cell viability, increased the number of TUNEL-positive cells, and promoted internucleosomal DNA fragmentation. Compared to wild-type cells, gelsolin-deficient SMC showed resistance to apoptosis induced by the inflammatory cytokines. These results suggest a mechanistic role for gelsolin cleavage during SMC apoptosis, a process implicated in vessel development as well as stability of atherosclerotic plaque.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by the development of benign tumors in multiple organs often causing serious neurologic impairment. To develop a reliable genetic test for TSC, two-dimensional electrophoresis with denaturing gradient gel electrophoresis (2D DGGE) has been developed to detect mutations in TSC1. The 23 exons of TSC1 were amplified using two rounds of PCR. In the first round, all coding regions of TSC1 were amplified in four fragments ranging in size from 7.4 kb to 9.9 kb. In the second round, 32 fragments representing 23 exons were amplified using primers designed to avoid overlapping fragments and with a GC clamp on one end to optimise melting characteristics. These exon fragments were then separated by size in the first dimension using a polyacrylamide gel, and by melting characteristics in the second dimension using a urea/formamide gradient to yield 32 distinct bands. If a mutation is present, four bands instead of one, are typically observed. During the development of this assay, we analysed 63 patient samples with known TSC1 mutations from prior studies. These 63 patients had 68 known mutations or polymorphisms. With DGGE, all 68 of these were identified (45 point mutations, 3 small insertions, 20 small deletions) and an additional 27 single base variants were discovered. To evaluate the assay, we analysed 19 of these samples in a blinded study. In the blinded analysis, 19/20 (95%) known mutations or polymorphisms were detected. The single missed mutation in the blinded analysis could be identified in retrospect and the assay was modified accordingly. During this study, we identified 2 new mutations (exon 8 and exon 15), a new polymorphism (intron 4), and the first variant identified in a non-coding exon (exon 2).

Tuberous sclerosis (TSC) is a genetic disorder that results in the development of hamartomatous lesions in a variety of organ systems. Both the prevalence of the disease and the often devastating consequences of these tumors pose a serious health and medical care problem. The disease has been mapped to two distinct genetic loci in humans, and although the genes (TSC1 and TSC2) for both loci have recently been cloned, their function remains an enigma. Data presented here demonstrates that TSC2 protein can bind and selectively modulate transcription mediated by members of the steroid receptor superfamily of genes. These data place TSC2 into a growing list of nuclear receptor coregulators and strengthen the expanding body of evidence that these coregulators may play critical roles in cellular differentiation.

A new member of the gelsolin/villin family of actin regulatory proteins was initially identified by screening an adult murine brain cDNA library with a probe for bovine adseverin. The predicted amino acid sequence of the 92 kDa murine protein p92 (advillin) is 75% homologous to villin and 65% homologous to gelsolin and adseverin. It shares a six domain structure with other gelsolin family members and has a carboxy-terminal headpiece, similar to, yet distinct from, villin. Northern blot analysis shows a high level of mRNA expression in murine uterus and human intestine. In situ mRNA analysis of adult murine tissues demonstrates that the message is most highly expressed in the endometrium of the uterus, the intestinal lining, and at the surface of the tongue. In murine embryonic development, strong expression of the message is observed by day 14.5 in dorsal root ganglia and trigeminal ganglia. Expression is also noted at day 16.5 in cerebral cortex. We propose that p92 (advillin) has unique functions in the morphogenesis of neuronal cells which form ganglia, and that it may compensate to explain the near normal phenotype observed in villin-deficient mice.

Angiomyolipomas can occur sporadically or in association with tuberous sclerosis complex (TSC). TSC is an autosomal dominant disorder characterized by seizures, mental retardation, and benign tumors of the brain, heart, kidney, and skin. Angiomyolipomas are more common in women than in men, suggesting a possible hormonal influence on tumor growth. In this study, 35 angiomyolipomas from 23 patients were immunostained with antibodies to estrogen receptor (ER) and progesterone receptor (PR). Eleven angiomyolipomas (31%) contained clusters of PR-immunoreactive smooth muscle cells. None contained ER-immunoreactive cells. Of the 21 tumors from patients with TSC, 11 (48%) were PR immunoreactive. All of the PR-immunoreactive angiomyolipomas were from women younger than 50 years of age, and all except one of these women had TSC. This study suggests that hormonal factors play a role in the pathogenesis of TSC-associated angiomyolipomas.