Cytoplasmic localization of PML particles in laminopathies.

There is growing evidence that laminopathies, diseases associated with mutations in the LMNA gene, are caused by a combination of mechanical and gene regulatory distortions. Strikingly, there is a large variability in disease symptoms between individual patients carrying an identical LMNA mutation. This is why classical genetic screens for mutations appear to have limited predictive value for disease development. Recently, the widespread occurrence of repetitive nuclear ruptures has been described in fibroblast cultures from various laminopathy patients. Since this phenomenon was strongly correlated with disease severity, the identification of biomarkers that report on these rupture events could have diagnostic relevance. One such candidate marker is the PML nuclear body, a structure that is normally confined to the nuclear interior, but leaks out of the nucleus upon nuclear rupture. Here, we show that a variety of laminopathies shows the presence of these cytoplasmic PML particles (PML CPs), and that the amount of these protein aggregates increases with severity of the disease. In addition, between clinically healthy individuals, carrying LMNA mutations, significant differences can be found. Therefore, we postulate that detection of PML CPs in patient fibroblasts could become a valuable marker for diagnosis of disease development.

Disturbed nuclear orientation and cellular migration in A-type lamin deficient cells.

The nuclear lamina and the cytoskeleton form an integrated structure that warrants proper mechanical functioning of cells. We have studied the correlation between structural alterations and migrational behaviour in fibroblasts with and without A-type lamins. We show that loss of A-type lamins causes loss of emerin and nesprin-3 from the nuclear envelope, concurring with a disturbance in the connection between the nucleus and the cytoskeleton in A-type lamin-deficient (lmna -/-) cells. In these cells functional migration assays during in vitro wound healing revealed a delayed reorientation of the nucleus and the microtubule-organizing center during migration, as well as a loss of nuclear oscillatory rotation. These observations in fibroblasts isolated from lmna knockout mice were confirmed in a 3T3 cell line with stable reduction of lmna expression due to RNAi approach. Our results indicate that A-type lamins play a key role in maintaining directional movement governed by the cytoskeleton, and that the loss of these karyoskeletal proteins has important consequences for functioning of the cell as a mechanical entity.

The caspase-9 derived C-terminal fragment of cytokeratin 18 modulates topoisomerase action.

During early apoptosis the 33 amino acid C-terminal cytokeratin 18 (CK18) fragment is released by caspase-9 cleavage at the 393DALD/S site. This basic peptide relocates from the cytoskeleton to the nucleoplasm as shown by confocal laser scanning. It is shown that the C-terminal peptide modulates topoisomerase activity as measured by relaxation of plasmid DNA. In an in vitro assay recombinant caspase-induced chromatin condensation is inhibited by the peptide and at the electron microscopical level a clear inhibition of nucleolar breakdown was observed in its presence. We hypothesize that the C-terminal CK18 fragment exerts an effect in the nucleolus by stimulating rRNA transcription and processing via modulation of enzymatic activity of topoisomerase I. This leads to preservation of general transcriptional activity required to exert active steps during early stages of programmed cell death.

Smoothelin, a novel cytoskeletal protein specific for smooth muscle cells.

The characterization of a novel 59-kD cytoskeletal protein is described. It is exclusively observed in smooth muscle cells by Northern blotting and immunohistochemical analysis and therefore designated "smoothelin." A human smooth muscle cDNA library was screened with the monoclonal antibody R4A, and a full-size cDNA of the protein was selected. The cDNA was sequenced and appeared to contain a 1,113-bp open reading frame. Based on the cDNA sequence, the calculated molecular weight of the polypeptide was 40 kD and it was demonstrated to contain two N-glycosylation sites. Computer assisted analysis at the protein level revealed a 56-amino acid domain with homologies of approximately 40% with a sequence bordering the actin-binding domains of dystrophin, utrophin, beta-spectrin and alpha-actinin. In situ hybridization demonstrated that human smoothelin is encoded by a single copy gene which is located on chromosome 22. Immunohistochemistry and Western blotting revealed synthesis of smoothelin in smooth muscle of species evolutionarily as far apart as human and teleost. Northern blotting indicated that sequence as well as size of the mRNA (approximately 1,500 bases) are conserved among vertebrates. Cell fractionation studies and differential centrifugation showed that the protein cannot be extracted with Triton X-100, which indicates that it is a part of the cytoskeleton. Transfection of the human cDNA into smooth muscle cells and COS7 cells produced a protein of 59 kD, which assembled into a filamentous network. However, in rat heart-derived myoblasts association with stress fibers was most prominent. Smoothelin was not detected in primary or long term smooth muscle cell cultures. Also, transcription of smoothelin mRNA was almost instantly halted in smooth muscle tissue explants. We conclude that smoothelin is a new cytoskeletal protein that is only found in contractile smooth muscle cells and does not belong to one of the classes of structural proteins presently known.

Assembly of amino-terminally deleted desmin in vimentin-free cells.

To study the role of the amino-terminal domain of the desmin subunit in intermediate filament (IF) formation, several deletions in the sequence encoding this domain were made. The deleted hamster desmin genes were fused to the RSV promoter. Expression of such constructs in vimentin-free MCF-7 cells as well as in vimentin-containing HeLa cells, resulted in the synthesis of mutant proteins of the expected size. Single- and double-label immunofluorescence assays of transfected cells showed that in the absence of vimentin, desmin subunits missing amino acids 4-13 are still capable of filament formation, although in addition to filaments large numbers of desmin dots are present. Mutant desmin subunits missing larger portions of their amino terminus cannot form filaments on their own. It may be concluded that the amino-terminal region comprising amino acids 7-17 contains residues indispensable for desmin filament formation in vivo. Furthermore it was shown that the endogenous vimentin IF network in HeLa cells masks the effects of mutant desmin on IF assembly. Intact and mutant desmin colocalized completely with endogenous vimentin in HeLa cells. Surprisingly, in these cells endogenous keratin also seemed to colocalize with endogenous vimentin, even if the endogenous vimentin filaments were disturbed after expression of some of the mutant desmin proteins. In MCF-7 cells some overlap between endogenous keratin and intact exogenous desmin filaments was also observed, but mutant desmin proteins did not affect the keratin IF structures. In the absence of vimentin networks (MCF-7 cells), the initiation of desmin filament formation seems to start on the preexisting keratin filaments. However, in the presence of vimentin (HeLa cells) a gradual integration of desmin in the preexisting vimentin filaments apparently takes place.

Transgenic expression of the muscle-specific intermediate filament protein desmin in nonmuscle cells.

The coding region of the hamster desmin gene was fused to the 5' flanking sequences of the hamster vimentin gene and introduced into the germ line of mice. The expression of this intermediate filament gene construct (pVDes) was analyzed at the RNA and protein level in transgenic mice as well as in fibroblast cell lines and primary hepatocyte cultures derived from these mice. In all transgenic mice, the pVDes-encoded protein was coexpressed with mouse vimentin in a tissue-specific fashion and was indistinguishable from normal hamster desmin. Culturing of transgenic hepatocytes induced desmin expression indicating that 3.2 kbp of the vimentin gene 5' region regulates both tissue-specific and tissue culture-induced intermediate filament protein expression. Immunohistochemical staining and double-label immunoelectron microscopy of cultured transgenic fibroblasts showed that the pVDes protein assembled into intermediate filaments which colocalized with the mouse vimentin filaments. Endogenous vimentin RNA levels were not influenced by high-level pVDes expression. The coexpression of desmin and vimentin in nonmuscle cells did not result in detectable developmental, morphological, or physiological abnormalities.

Molecular alterations during insulinoma tumorigenesis.

Insulinomas are the most common functioning endocrine pancreatic tumors (EPTs). They present with clinical symptoms as a consequence of hypoglycemia induced by inappropriate insulin secretion. The etiology of these tumors is poorly understood. Some tumors may harbor MEN1 gene mutations, the susceptibility gene of the multiple endocrine neoplasia type I syndrome, but most cases show wildtype MEN1. Currently, no reliable clinical tests are available to differentiate benign from malignant tumors. Approximately 30% of the tumors are unresectable, and they often show different growth rates, which hampers treatment. Therefore, a better understanding of the molecular processes underlying the development and progression of insulinomas is required to improve diagnosis, prognosis and therapy. Here we summarize the progress that has been made in insulinoma research in the past decade. We describe the clinical detection, classification and treatment of these tumors, and review the multiplicity of molecular and genetic studies that investigated tumor development and progression using either primary tumors, transgenic mouse models or tumor-derived cell lines. The identification of many interactors of the MEN1 gene product menin, as well as recurrent chromosomal abnormalities that pinpoint candidate genes of interest will likely result in a better understanding of the molecular pathways involved in insulinoma tumorigenesis. In addition, these studies will pave the way for the identification of novel targets for therapeutical intervention and more reliable markers for clinical diagnosis and prognosis.

Role of nuclear lamina-cytoskeleton interactions in the maintenance of cellular strength.

The response of individual cells to cellular stress is vital for cellular functioning. A large network of physically interconnected cellular components, starting from the structural components of the cells' nucleus, via cytoskeleton filaments to adhesion molecules and the extracellular matrix, constitutes an integrated matrix that functions as a scaffold allowing the cell to cope with mechanical stress. Next to a role in mechanical properties, this network also has a mechanotransductional function in the response to mechanical stress. This signaling route does not only regulate a rapid reorganization of structural components such as actin filaments, but also stimulates for example gene activation via NFkappaB and other transcription factors. The importance of an intact mechano-signaling network is illustrated by the physiological consequences of several genetic defects of cellular network components e.g. actin, dystrophin, desmin and lamins. These give rise to an impaired response of the affected cells to mechanical stress and often result in dystrophy of the affected tissue. Recently, the importance of the cell nucleus in cellular strength has been established. Several new interconnecting proteins, such as the nesprins that link the nuclear lamina to the cytoskeleton, have been identified. Furthermore, the function of nuclear lamins in determining cellular strength and nuclear stability was illustrated in lamin-knock-out cells. Absence of the A-type lamins or mutations in these structural components of the nuclear lamina lead to an impaired cellular response to mechanical stress and disturbances in cytoskeletal organization. In addition, laminopathies show clinical phenotypes comparable to those seen for diseases resulting from genetic defects in cytoskeletal components, further indicating that lamins play a central role in maintaining the mechanical properties of the cell.

Marked differences in survival rate between smokers and nonsmokers with HPV 16-associated tonsillar carcinomas.

Oncogenic human papillomavirus (HPV) is a causative agent in a subgroup of head and neck carcinomas, particularly tonsillar squamous cell carcinomas (TSCC). This study was undertaken because controversial data exist on the physical status of HPV-DNA and the use of p16(INK4A) overexpression as surrogate HPV marker, and to examine the impact of HPV and tobacco consumption on the clinical course of TSCC. Tissue sections of 81 TSCC were analyzed by HPV 16-specific fluorescence in situ hybridization (FISH) and p16(INK4A)-specific immunohistochemistry. Results were correlated with clinical and demographic data. HPV 16 integration was detected by FISH as punctate signals in 33 out of 81 (41%) TSCC, 32 of which showed p16(INK4A) accumulation. Only 5 out of 48 HPV-negative tumors showed p16(INK4A) immunostaining (p < 0.0001). The presence of HPV furthermore correlates significantly with low tobacco (p = 0.002) and alcohol intake (p = 0.011), poor differentiation grade (p = 0.019), small tumor size (p = 0.024), presence of a local metastasis (p = 0.001) and a decreased (loco)regional recurrence rate (p = 0.039). Statistical analysis revealed that smoking significantly increases the risk of cancer death from TSCC and that non-smoking patients with HPV-containing TSCC show a remarkably better disease-specific survival rate. HPV 16 is integrated in 41% of TSCC and strongly correlates with p16(INK4A) overexpression, implicating the latter to be a reliable HPV biomarker. Patients with HPV-positive tumors show a favorable prognosis as compared to those with HPV-negative tumors, but tobacco use is the strongest prognostic indicator. These findings indicate that oncogenic processes in the tonsils of non-smokers differ from those occurring in smokers, the former being related to HPV 16 infection.

DNA copy number status is a powerful predictor of poor survival in endocrine pancreatic tumor patients.

The clinical behavior of endocrine pancreatic tumors (EPTs) is difficult to predict in the absence of metastases or invasion to adjacent organs. Several markers have been indicated as potential predictors of metastatic disease, such as tumor size > or =2 cm, Ki67 proliferative index > or =2%, cytokeratin (CK) 19 status, and recently in insulinomas, chromosomal instability (CIN). The goal of this study was to evaluate the value of these markers, and in particular of the CIN, to predict tumor recurrence or progression and tumor-specific death, using a series of 47 insulinomas and 24 non-insulinoma EPTs. From these EPT cases, a genomic profile has been generated and follow-up data have been obtained. The proliferative index has been determined in 68 tumors and a CK19 expression pattern in 50 tumors. Results are statistically analyzed using Kaplan-Meier plots and the log-rank statistic. General CIN, as well as specific chromosomal alterations such as 3p and 6q loss and 12q gain, turned out to be the most powerful indicators for poor tumor-free survival (P< or =0.0004) and tumor-specific death (P< or =0.0113) in insulinomas. The CIN, chromosome 7q gain, and a proliferative index > or =2% were reliable in predicting a poor tumor-free survival in non-insulinoma EPTs (P< or =0.0181, whereas CK19 expression was the most optimal predictor of tumor-specific death in these tumors. In conclusion, DNA copy number status is the most sensitive and efficient marker of adverse clinical outcome in insulinomas and of potential interest in non-insulinoma EPTs. As a consequence, this marker should be considered as a prognosticator to improve clinical diagnosis, most practically as a simple multi-target test.

Intermediate filaments in malignant melanomas. Identification and use as marker in surgical pathology.

Intermediate-sized filaments have been studied in human malignant melanomas and in normal melanocytes by immunofluorescence microscopy with antibodies directed against keratin, vimentin, desmin, neurofilament protein, and glial filament protein. Both human melanotic and amelanotic tumor cells and tumor metastases as well as normal melanocytes in human skin and in the rat eye contain exclusively intermediate filaments of the vimentin type. No reaction was seen with antibodies to keratin, desmin, neurofilaments, or glial filaments. These latter four antisera, however, gave strong reactions in epidermis and other epithelial tissues, muscle, or neural tissues, respectively. The results favor a mesenchymal character of melanocytes, although a neuroectodermal origin in an early developmental stage is possible. The finding that melanomas contain exclusively vimentin intermediate filaments may prove useful in differential diagnosis of melanomas from other tumor types.

The nuclear envelope, a key structure in cellular integrity and gene expression.

The envelope that encapsulates the cell nucleus has recently gained considerable interest, as several clinical syndromes are linked to mutations in its molecular components. Most disorders recognized so far are caused by defects in the nuclear lamins, building blocks of a filamentous network lining the nucleoplasmic side of the inner nuclear membrane. Nuclear lamins are the evolutionary precursors of cytoskeletal intermediate filaments and associate in a head-to-tail manner into a stable lamina at the nuclear periphery and into a more dispersed structure in the nucleoplasm. Lamins have a scaffolding function for several nuclear processes such as transcription, chromatin organization and DNA replication, and maintain nuclear and cellular integrity. Mutations in the LMNA gene, encoding A-type lamins, can cause cardiac and skeletal muscle disease, lipodystrophy and premature ageing phenotypes. Hence, the integrity of the nuclear envelope seems essential for longevity. Furthermore, the laminopathies provide evidence that metabolism and ageing are as tightly linked in humans as they are in model organisms such as C. elegans. In this review, we elaborate on the structure and functions of nuclear lamins, the spectrum of syndromes related to mutations in nuclear envelope components and pathogenic concepts unifying these disorders.

Investigating the race for the surface and skin integration in clinically retrieved abutments with two-photon microscopy.

Bone conduction hearing implants can rehabilitate some types of hearing loss. A hydroxyapatite (HA)-coated skin-penetrating abutment was developed to allow for soft tissue preservation and increased skin-abutment adherence. Inflammation is thought to relate to bacterial infection of pockets around the abutment. Upon integration, the host's ability to cover the abutment surface ("race for the surface"), and thus control and prevent competitive bacteria from colonizing it, is improved. However, the attachment mechanisms behind it are not clear. In this study, we applied two-photon microscopy to visualize tissue attachment on abutments retrieved from patients. Skin integration markers were validated and applied to four HA-coated abutments. Evidence of skin integration was found, including the presence of hemidesmosomes, a basement membrane, dermal collagen and vascularization. Cases with clinical signs of severe inflammation and evident biofilm formation showed limited skin integration based on these indicators, confirming the applicability of the "race for the surface" model.

Molecular parameters associated with insulinoma progression: chromosomal instability versus p53 and CK19 status.

Insulinomas represent the predominant syndromic subtype of endocrine pancreatic tumors (EPTs). Their metastatic potential cannot be predicted reliably using histopathological criteria. In the past few years, several attempts have been made to identify prognostic markers, among them TP53 mutations and immunostaining of p53 and recently cytokeratin 19 (CK19). In a previous study using conventional comparative genomic hybridization (CGH) we have shown that chromosomal instability (CIN) is associated with metastatic disease in insulinomas. It was our aim to evaluate these potential parameters in a single study. For the determination of CIN, we applied CGH to microarrays because it allows a high-resolution detection of DNA copy number changes in comparison with conventional CGH as well as the analysis of chromosomal regions close to the centromeres and telomeres, and at 1pter-->p32, 16p, 19 and 22. These regions are usually excluded from conventional CGH analysis, because they may show DNA gains in negative control hybridizations. Array CGH analysis of 30 insulinomas (15 tumors of benign, eight tumors of uncertain and seven tumors of malignant behavior) revealed that >or=20 chromosomal alterations and >or=6 telomeric losses were the best predictors of malignant progression. A subset of 22 insulinomas was further investigated for TP53 exon 5-8 gene mutations, and p53 and CK19 expression. Only one malignant tumor was shown to harbor an arginine 273 serine mutation and immunopositivity for p53. CK19 immunopositivity was detected in three malignant tumors and one tumor with uncertain behavior. In conclusion, our results indicate that CIN as well as telomeric loss are very powerful indicators for malignant progression in sporadic insulinomas. Our data do not support a critical role for p53 and CK19 as molecular parameters for this purpose.

Interphase cytogenetics of hematological cancer: comparison of classical karyotyping and in situ hybridization using a panel of eleven chromosome specific DNA probes.

Numerical chromosome aberrations were detected in hematological cancers by nonradioactive in situ hybridization (ISH) procedures, using centromere specific probes for chromosomes 1, 7, 8, 9, 10, 11, 16, 17, 18, X, and Y. All 15 cases could be evaluated by ISH for these 11 probes. Our experiments show that in seven of these randomly selected leukemia bone marrow cell suspensions numerical aberrations for one or two chromosomes could be detected by this method. The results of ISH on interphase nuclei and in some cases on metaphase preparations were compared with karyotyping data. Seven cases of chromosomal aberrations observed with ISH (three for monosomy and four for trisomy) were confirmed by this classical cytogenetic technique, whereas in five instances an aberration was found only with ISH (twice for monosomy, twice for trisomy, and one disomy for the Y-probe). One case of a trisomy for chromosome 1 observed by ISH on interphase nuclei could be explained by a marker chromosome, a finding that was further substantiated by ISH on metaphase spreads. In this case double-target ISH on interphase cells with the probes for chromosomes 1 and 16 strongly suggested a translocation between these chromosomes. Also, in one case a marker chromosome could be characterized as a translocation between chromosomes 7 and 17. In this latter case the cytogenetic examinations revealed only monosomy for chromosomes 7 and 17 in addition to noncharacterized marker chromosomes. Our results indicate that the nonradioactive ISH procedure in combination with chromosome specific repetitive centromeric probes is a powerful tool for studying both numerical and structural chromosomal aberrations in interphase nuclei of leukemias. It may therefore become a valuable and routine diagnostic tool in addition to the existing karyotyping procedures.

Numerical chromosome 1, 7, 9, and 11 aberrations in bladder cancer detected by in situ hybridization.

Forty transitional cell carcinomas of the human urinary bladder (TCCs) were examined for numerical aberrations of chromosomes 1, 7, 9, and 11 by in situ hybridization using chromosome-specific probes. Our interphase cytogenetic study of 24 low-grade, noninvasive TCCs, which were near-diploid by flow cytometry, showed a numerical aberration for at least 1 of these chromosomes in 14 of these cases. Most strikingly, a monosomy for chromosome 9 was found in 9 of 24 low-grade TCCs. A trisomy for chromosomes 1, 7, and 11 was detected in 5, 2, and 1 case(s), respectively. In 1 case a monosomy for chromosome 1 was detected by in situ hybridization. Monosomy for chromosome 9 was the only detected numerical change in 5 low-grade TCC cases. Examination of 16 invasive TCCs showed extra copies for chromosomes 1 and 7 in 7 flow cytometrically diploid cases with numerical chromosome aberrations; also, loss of chromosome 9 was detected. In 5 invasive and 2 noninvasive aneuploid/tetraploid TCCs a profound imbalance between the different chromosomes was found. In 5 of these cases an evident underrepresentation of chromosome 9 in comparison to chromosomes 1, 7, and 11 was detected. This underrepresentation of chromosome 9 in diploid, as well as aneuploid, TCCs, and in some cases the constant ratio between this chromosome and the other chromosomes, may be explained by a process of tetraploidization. Therefore, loss of chromosome 9 may be one of the primary genetic events in TCC oncogenesis, with secondary events, such as tetraploidization, correlated to tumor progression. Our results show that in situ hybridization can be routinely used to study important cytogenetic changes which occur during the development of a malignant disease.

Structural chromosome 1 aberrations in transitional cell carcinoma of the bladder: interphase cytogenetics combining a centromeric, telomeric, and library DNA probe.

Fluorescence in situ hybridization (FISH) was used to study numerical and structural chromosome 1 aberrations in interphase nuclei of transitional cell carcinomas (TCCs) of the urinary bladder. One of the characteristic numerical aberrations, as detected previously in low-grade noninvasive TCCs, included trisomy for chromosome 1 (A. H. N. Hopman et al., Cancer Res., 51: 644-651, 1991). We examined in more detail 22 cases with a centromeric (1q12) and a telomeric associated (1p36) DNA probe and with a library DNA probe from sorted human chromosome 1 in single- and double-target FISH procedures. All flow cytometrically determined DNA diploid TCCs (13 cases), which showed three spots for 1q12 (6 cases), had two spots for 1p36. Since the library DNA probe showed three separate domains in the nuclei of these cases, the additional copy for 1q12 could be explained as an extra chromosome 1p-, containing the 1q12 target. In the flow cytometrically determined DNA tetraploid/aneuploid tumors, the results were more complex. In 6 of 9 cases, we observed an overrepresentation of 1q12 as compared to 1p36, also suggesting the presence of extra copies of 1p- chromosomes. The results of the present study demonstrate the utility of the FISH method to assess structural chromosome aberrations in interphase nuclei of solid tumors.

Distinct nonrandom patterns of chromosomal aberrations in the progression of squamous cell carcinomas of the head and neck.

Fifty-one randomly selected primary squamous cell carcinomas of the head and neck, derived from the larynx (n = 18) and pharynx (oropharynx, n = 18, and hypopharynx, n = 15) were analyzed with centromeric probes for chromosomes 1, 7, 9, 11, 17, and 18 to study numerical aberrations, chromosome imbalances, and aneuploidy by fluorescence in situ hybridization. The tumors were grouped into nonmetastasizing (N0) tumors (from patients clinically free of lymph node metastases for at least 18 months after surgery, n = 25) and metastasizing (N1-3) tumors (n = 26). We found a significant association between the tumor ploidy and the nodal status; in the N0 group, diploidy prevailed, and the most common aberration was loss to monosomy for chromosome 9 (44%), whereas in the N1-3 group, aneuploidy predominated (P = 0.002). Specifically, these genomic changes associated with progression to metastasis were: (a) tetrasomic or polysomic chromosomes were detected in 17 of 26 N1-3 tumors but in none of the 25 N0 tumors (P < 0.0001); (b) heterogeneous chromosomal copy numbers (i.e., extensive chromosomal imbalances) were also much more frequent in the N1-3 tumors (69.2% versus 24.0% in the N0 group; P = 0.018); and (c) loss of chromosome 9 (73%) and gains of chromosomes 7 (35%) and 17 (31%) persisted, but in addition, loss of chromosome 18 occurred in 31%. Overexpression of the p53 protein correlated with an increased incidence of chromosomal imbalances and aneuploidy (P < 0.001) and, hence, constituted an additional risk factor. The lower metastatic potential of larynx tumors as compared with tumors from the pharynx was reflected by a lower incidence of these genomic changes. These specific patterns of chromosomal aberrations can characterize and distinguish different stages of tumor progression of squamous cell carcinomas of the head and neck and should be valuable diagnostic and prognostic markers.

NSP-encoded reticulons are neuroendocrine markers of a novel category in human lung cancer diagnosis.

The NSP gene was recently shown to constitute the prototype of a novel gene family, to be selectively transcribed in neural and endocrine cells, and to encode three overlapping proteins, NSP-A, NSP-B, and NSP-C. These proteins were collectively designated reticulons, because they were found to be anchored to membranes of the endoplasmic reticulum through their common carboxy-terminal regions. The goal of the present study was to determine whether the reticulons might be used as markers for neuroendocrine differentiation in human lung tumors. Therefore, the tissue distribution of the NSP-A protein was studied and expression in human lung tumors was evaluated. Immunohistochemical analysis of normal tissues with monoclonal antibodies specifically recognizing the NSP-A protein indicated that NSP-A exhibits a distinct neuroendocrine distribution pattern since it was found to be expressed in a variety of cells with an established neuroendocrine phenotype but not in cells lacking such features. Results with specimens of a wide variety of primary human tumors provided further support for this claim. Immunohistochemical analysis of primary lung carcinomas revealed that NSP-A was readily detectable in small cell lung carcinoma (SCLCs) (8 of 12) and carcinoid tumors of the lung (3 of 3) but not in nonneuroendocrine non-SCLCs (0 of 10). In 13 of 27 non-SCLCs expressing the neural cell adhesion molecule and/or neurofilament proteins, however, NSP-A was found to be expressed. Northern blot analysis of human lung carcinoma cell lines revealed expression of NSP-A- and/or NSP-C-encoding mRNAs in all 18 SCLC cell lines that were studied, except one; however, no expression of these mRNAs could be detected in any of the 11 non-SCLC cell lines tested. The NSP transcript encoding NSP-B was found only in SCLC cell line NCI-H82. In conclusion, the results of our studies suggest that, in lung tumor cells, expression of NSP-A and most likely also NSP-C is restricted to cells with a neuroendocrine phenotype.

Colocalization of basal and luminal cell-type cytokeratins in human prostate cancer.

In the epithelium of secretory acini of the prostate two different cell types can be discriminated on the basis of localization, morphology, and degree of differentiation, the luminal and basal cells. The possibility of a developmental relationship between basal and luminal cells has been a subject of interest in several studies. According to the stem cell model at least three cell types, i.e., stem, amplifying, and transit cells, can be discriminated in the epithelium of prostate secretory acini. We previously reported that in the process of degeneration and regeneration in normal rat prostate a population of cells could be identified as candidates for the amplifying cells. These cells showed a keratin expression profile intermediate between those of basal and luminal cells. We now show, by using keratin antibodies, that also in normal human prostate at least three subpopulations of cells can be identified, one of them putatively representing amplifying cells as defined in the stem cell model. Furthermore, these antibodies were used to obtain a better insight into the different cell types involved in the etiology and progression of prostatic carcinoma. Both primary and hormone-independent prostatic tumors were investigated. Our results indicated that the candidate stem cell population was absent in prostatic carcinoma. Unlike earlier reports on the unique presence of cells with luminal characteristics in prostatic carcinoma, we identified also a population of cells coexpressing basal and luminal cell-type cytokeratins in primary and hormone-independent prostatic carcinoma. Since amplifying cells are defined in the stem cell model as precursors of transit (luminal) cells in the hierarchical pathway of prostatic epithelium differentiation, we postulate that on the basis of the keratin expression profile this subpopulation is most likely the target for neoplastic transformation.