The clinicopathologic, immunohistochemical, and ultrastructural features of soft tissue angiosarcomas are not well defined. Eighty cases of angiosarcoma that involved the deep subcutis, skeletal muscle, retroperitoneum, mesentery, and mediastinum are reported. The lesions occurred in 50 male and 30 female patients who were 5-97 years of age; the peak incidence was in the seventh decade of life. A variety of associated conditions were documented in 20 of these cases, including a history of other neoplasms (some irradiated), synthetic vessel grafts, heritable conditions, and prior trauma or surgery. The angiosarcomas occurred in the extremities (n = 43 cases), trunk (n = 28), and the head and neck (n = 9) regions, with the thigh and the retroperitoneum being the most common sites. They often were characterized as enlarging, painful masses of several weeks' duration and were occasionally associated with acute hemorrhage, anemia, or a coagulopathy. The tumors measured 1-15 cm in diameter (median 5 cm) and frequently were hemorrhagic and multinodular. There was a wide morphologic spectrum within and between cases, including areas similar to cavernous and capillary hemangioma, Dabska tumor, spindle cell and epithelioid hemangioendothelioma, various spindle cell sarcomas, or carcinoma. Histologically, epithelioid angiosarcoma was the most frequently observed pattern; 70% of cases had epithelioid cells that were arranged in nests, clusters, papillae, and gaping vascular channels. Hemorrhage tended to obscure the diagnosis in several cases and often was associated with papillary endothelial hyperplasia-like areas. All 42 cases studied immunohistochemically stained at least focally for Factor VIII-related antigen, and nearly all stained strongly for vimentin, which accentuated the endothelial cells and vessel lumen formation. CD34 antigen was detected in 74% of cases, BNH9 in 72%, and cytokeratins in 35%. Epithelial membrane antigen, S-100 protein, and HMB45 were not detected. Fifty-five percent of the tumors had intracytoplasmic aggregates of laminin. Immunostains for alpha-smooth muscle actin demonstrated a prominent pericytic component in several tumors (24%). Ki67 immunostains with MIB1 indicated high proliferative activity >> or =10%) in 72% of cases. p53 immunoreactivity (>20% nuclear staining) was observed in 20% of cases. Ultrastructural studies performed on poorly differentiated areas of 12 cases showed groups of cells, which were frequently epithelioid, surrounded by basal lamina, and closely associated with pericytes, along with intercellular and intracellular lumina with or without red blood cells. Whorls of abundant intermediate filaments, occasional tonofilamentlike structures, and pinocytotic vesicles also were noted. In contrast to the findings of others, Weibel-Palade bodies were not seen. Follow-up in 49 cases (61%) showed that 53% of patients were dead of disease at a median interval of 11 months, whereas 31% had no evidence of disease at a median interval of 46 months. The remaining patients were either alive with disease (14%) or alive but disease status was unknown (2%). There were local recurrences in 20% of cases and distant metastases in 49%, most frequently to the lungs, followed by the lymph nodes, soft tissues, bone, liver, and other sites. These results indicate that angiosarcoma of soft tissue is a high-grade sarcoma. Older patient age, tumor location in the retroperitoneum, and larger tumor size as well as detection of MIB1 in>> or =10% of the tumor cell population were all associated with a poorer prognosis.

Effects of regulatory proteins on the actomyosin subfragment 1 (acto-S1) Mg2+-ATPase activity were studied over a range of S1 concentration at low actin concentration such that the specific activity was constant in the absence of the regulatory proteins. In their presence, the activity was inhibited at low [S1] and activated at higher [S1] in each of three cases: 1) troponin + tropomyosin + Ca2+; 2) tropomyosin; 3) troponin + tropomyosin - Ca2+. The [S1] required to activate increased in the order 1, 2, 3. In all three cases, however, the inhibition increased toward 100% when the [S1] approached zero. Tropomyosin titrations of acto-S1 ATPase at low [S1] which resulted in inhibition were hyperbolic and gave a binding constant of K approximately 10(7) M-1. In contrast, at higher [S1], tropomyosin titrations were sigmoidal, indicating cooperative effects on activation. These results suggest a modification of the simple steric blocking theory of regulation, in which it is postulated that both of the Ca2+-dependent positions of tropomyosin on the thin filament block the formation of active acto-S1-nucleotide intermediates at low [S1], and in which tropomyosin occupies a third "nonblocking" position in the active state at high [S1]. In this modified model, Ca2+ facilitates the binding of myosin heads, leading to the active site.

A monoclonal antibody to vimentin (RBA1) and a polyclonal antiserum to glial fibrillary acidic protein (GFAP) were used in double labeling experiments to examine astrocyte intermediate filaments in development and wounding. RBA1 bound to radial glia in newborn rat parietal cortex that are predominantly anti-GFAP-negative. The RBA1-positive radial fibers disappeared by postnatal day 20 with the greatest rate of disappearance occurring between day 8 and day 15. Between birth and day 20, the anti-GFAP staining increased to the adult pattern in mature shaped astrocytes. Some overlay was observed between the binding patterns of the two antibodies. Stab wounds to cortical areas were made at a developmental time when there were normally no RBA1-positive astrocytes. RBA1-positivity was present in some astrocytes but only at the edges of the wounds. The distribution patterns of RBA1-positive cells led to hypotheses concerning the possible function of vimentin in astrocytes and its regulation during development and wounding.

The intermediate filament cytoskeleton of cultured bovine kidney epithelial cells and human HeLa cells changes dramatically during mitosis. The bundles of cytokeratin and vimentin filaments progressively unravel into protofilament-like threads of 2-4 nm diameter, and intermediate filament protein is included in numerous, variously sized (0.2-1.5 microns) spheroidal aggregates containing densely stained granular particles of 5-16 nm diameter. We describe these mitotic bodies in intact cells and in isolated cytoskeletons. In metaphase to anaphase of normal mitosis and after colcemid arrest of mitotic stages, many cells contain all their detectable cytokeratin and vimentin material in the form of such spheroidal aggregate bodies, whereas in other mitotic cells such bodies occur simultaneously with bundles of residual intermediate filaments. In telophase, the extended normal arrays of intermediate filament bundles are gradually reestablished. We find that vimentin and cytokeratins can be organized in structures other than intermediate filaments. Thus, at least during mitosis of some cell types, factors occur that promote unraveling of intermediate filaments into protofilament-like threads and organization of intermediate filament proteins into distinct granules that form large aggregate bodies. Some cells, at least certain epithelial and carcinoma cells, may contain factors effective in structural modulation and reorganization of intermediate filaments.

The appearance of neurofilaments (NFs) and vimentin (Vim) in the nervous system of the mouse embryo was documented using immunohistochemical techniques. The three NF protein subunits appear early and simultaneously in central and peripheral neurons at 9 to 10 days of gestation. The onset of NF expression is concomitant with axon elongation and correlates extremely well with neurofibrillar differentiation and, in the case of autonomic ganglia, with the expression of adrenergic neurotransmitter properties. In the central and peripheral nervous system, NF expression is preceded by that of Vim, and both types of intermediate filaments coexist within the same cell for a short period of time.

In eukaryotic cells, microtubules, actin, and intermediate filaments interact to form the cytoskeletal network involved in determination of cell architecture, intracellular transport, modulation of surface receptors, mitosis, cell motility, and differentiation. Cytoskeletal organization and dynamics depend on protein self-associations and interactions with regulatory elements such as microtubule-associated proteins (MAPs). The MAP family includes large proteins like MAP-1A, MAP-1B, MAP-1C, MAP-2, and MAP-4 and smaller components like tau and MAP-2C. This review focuses on relevant aspects of MAP function, with emphasis on their roles in modulating cytoskeletal interactions. In this context, MAP expression mechanisms and posttranslational modifications are also discussed. Microtubule-associated proteins have a rather widespread distribution among cells, but certain MAPs have been identified in specific cell types. Within single neurons, MAP-2 is dendritic while tau is preferentially an axonal protein. Their expression is developmentally regulated. Even though MAPs share a capacity to interact with the COOH-terminal tubulin domain, stabilize microtubules, and link them with other cytoskeletal polymers, they exhibit structural differences. However, MAP-2, MAP-4, and tau have common repetitive microtubule-binding motifs. Microtubule-associated proteins not only control cytoskeletal integrity, but they also appear to interact with highly structural elements of cells. Molecular biological approaches permitted localization of new MAPs in cultured mammalian cells and invertebrate organisms and other microtubule-interacting proteins that exhibit transient interactions with microtubules. The structural/functional aspects of several new MAP-like proteins in centrosomes and the mitotic spindle, functionally implicated in cell cycle events, are also analyzed.

A 230,000 dalton polypeptide co-purifies through cycles of depolymerization and polymerization with the intermediate filament subunits, desmin and vimentin, from avian smooth muscle. This protein is also present in skeletal muscle and is distinct from myosin and filamin. Double immunofluorescence microscopy of cultured cells, using antisera shown to be specific by immunoautoradiography, has revealed that this protein has the same spatial distribution as desmin and vimentin. During skeletal myogenesis, all three antigens exist initially in multinucleate myotubes as wavy filaments throughout the cytoplasm. Within a week after myoblast fusion, they begin to coalesce at the peripheries of the myofibril Z discs, thereby attaining the distribution observed in mature muscle, a network of interlinked rings within the Z plane. Treatment of cultured myotubes with colcemid causes the filamentous forms of these three proteins to co-aggregate into cytoplasmic bundles, but has little effect on them when they are associated with the Z discs. Extraction of cells with nonionic detergent and high salt leaves cytoskeletons containing desmin, vimentin and the 230,000 dalton polypeptide with immunofluorescent patterns that are indistinguishable from one another. These data suggest that this high molecular weight protein is closely associated with desmin and vimentin filaments in muscle cells; to indicate this, we have named the protein synemin, from the Greek oa uv (with) and v eta mu alpha (filament).

Epidermal keratinocytes undergo a unique form of terminal differentiation and programmed cell death known as cornification. Cornification leads to the formation of the outermost skin barrier, i.e. the cornified layer, as well as to the formation of hair and nails. Different genes are expressed in coordinated waves to provide the structural and regulatory components of cornification. Differentiation-associated keratin intermediate filaments form a complex scaffold accumulating in the cytoplasm and, upon removal of cell organelles, fill the entire cell interior mainly to provide mechanical strength. In addition, a defined set of proteins is cross-linked by transglutamination in the cell periphery to form the so-called cornified envelope. Extracellular modifications include degradation of the tight linkages between corneocytes by excreted proteases, which allows corneocyte shedding by desquamation, and stacking and modification of the excreted lipids that fill the intercellular spaces between corneocytes to provide a water-repellant barrier. In hard skin appendages such as hair and nails these tight intercorneocyte connections remain permanent. Various lines of evidence exist for a role of organelle disintegration, proteases, nucleases, and transglutaminases contributing to the actual cell death event. However, many mechanistic aspects of kearatinocyte death during cornification remain elusive. Importantly, it has recently become clear that keratinocytes activate anti-apoptotic and anti-necroptotic pathways to prevent premature cell death during terminal differentiation. This review gives an overview of the current concept of cornification as a mode of programmed cell death and the anti-cell death mechanisms in the epidermis that secure epidermal homeostasis. This article is part of a Special Section entitled: Cell Death Pathways.

The expression of intermediate filament proteins is remarkably tissue-specific which suggests that the intermediate filament (IF) type(s) present in cells is somehow related to their biological function. However, in some cancers-particularly malignant melanoma and breast carcinoma, there is a strong indication that vimentin and keratin IFs are coexpressed, thus presenting as a dedifferentiated or interconverted (between epithelial and mesenchymal) phenotype. In this review, two in vitro models are presented which recapitulate the interconverted phenotype in human melanoma and breast carcinoma, and allow, for the first time, unique observations to be made with respect to the role of IFs in cancer progression. These studies have provided direct evidence linking overexpression of keratin IFs in human melanoma with increased migratory and invasive activity in vitro, which can be down-regulated by substituting dominant-negative keratin mutants. Overexpression of vimentin IFs in the breast carcinoma model leads to augmentation of motility and invasiveness in vitro, which can be transiently down-regulated by treatment with antisense oligonucleotides to vimentin. Additional experimental evidence suggests that the mechanism(s) responsible for the differential expression of metastatic properties associated with the interconverted phenotype rest(s) in the unique interaction, either direct or indirect, of IFs with specific integrins interacting with the extracellular matrix. In this review, we discuss the observations derived from the human melanoma and breast carcinoma models to address the hypothesis that the ability to coexpress vimentin and keratins confers a selective advantage to tumor cells in their interpretation of and response to signaling cues from the extracellular matrix. The ramifications of these observations are discussed with respect to the patholophysiology of the respective in situ tumors.

Recent studies have shown that premature cellular senescence and normal organ development and function depend on the type V intermediate filament proteins, the lamins, which are major structural proteins of the nucleus. This review presents an up-to-date summary of the literature describing new findings on lamin functions in various cellular processes and emphasizes the relationship between the lamins and devastating diseases ranging from premature aging to cancer. Recent insights into the structure and function of the A- and B- type lamins in normal cells and their dysfunctions in diseased cells are providing novel targets for the development of new diagnostic procedures and disease intervention. We summarize these recent findings, focusing on data from mice and humans, and highlight the expanding knowledge of these proteins in both healthy and diseased cells.

O-linked beta-N-acetylglucosamine (O-GlcNAc) is a highly dynamic intracellular protein modification responsive to stress, hormones, nutrients, and cell cycle stage. Alterations in O-GlcNAc addition or removal (cycling) impair cell cycle progression and cytokinesis, but the mechanisms are not well understood. Here, we demonstrate that the enzymes responsible for O-GlcNAc cycling, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are in a transient complex at M phase with the mitotic kinase Aurora B and protein phosphatase 1. OGT colocalized to the midbody during telophase with Aurora B. Furthermore, these proteins coprecipitated with each other in a late mitotic extract. The complex was stable under Aurora inhibition; however, the total cellular levels of O-GlcNAc were increased and the localization of OGT was decreased at the midbody after Aurora inhibition. Vimentin, an intermediate filament protein, is an M phase substrate for both Aurora B and OGT. Overexpression of OGT or OGA led to defects in mitotic phosphorylation on multiple sites, whereas OGT overexpression increased mitotic GlcNAcylation of vimentin. OGA inhibition caused a decrease in vimentin late mitotic phosphorylation but increased GlcNAcylation. Together, these data demonstrate that the O-GlcNAc cycling enzymes associate with kinases and phosphatases at M phase to regulate the posttranslational status of vimentin.

The intermediate filament protein Nestin identifies stem/progenitor cells in adult tissues, but the function of Nestin is poorly understood. We investigated Nestin expression and function in common lethal cancers. Nestin mRNA was detected in cell lines from small cell lung, and breast cancers, and particularly elevated in cell lines derived from prostate cancer metastases. Whereas the androgen-independent lines PC3, 22RV1, and DU145 all expressed Nestin transcripts under standard culture conditions, the androgen-dependent line LnCaP expressed Nestin only on androgen withdrawal. We confirmed associations of Nestin expression, androgen withdrawal, and metastatic potential by immunohistochemical analysis of samples from 254 prostate cancer patients. Cytoplasmic Nestin protein was readily identifiable in prostate cancer cells from 75% of patients with lethal androgen-independent disease, even in cancer sampled from the prostate itself. However, Nestin expression was undetectable in localized androgen-deprived tumors and in metastases without prior androgen deprivation. To address its function, we reduced Nestin levels with short hairpin RNAs, markedly inhibiting in vitro migration and invasion in prostate cancer cells but leaving cell growth intact. Nestin knockdown also diminished metastases 5-fold compared with controls despite uncompromised tumorigenicity at the site of inoculation. These results specify a function for Nestin in cell motility and identify a novel pathway for prostate cancer metastasis. Activity of this pathway may be selected by the extraprostatic environment or, as supported by our data, may originate within the prostate after androgen deprivation. Further dissection of this novel Nestin migration pathway may lead to strategies to prevent and neutralize metastatic spread.

Intermediate-sized filament proteins (IFP) are tissue specific in that antibodies to keratin, vimentin, desmin, glial fibrillary acidic protein (GFAP) and the neurofilament proteins can distinguish between cells of epithelial and mesenchymal origin as well as of myogenic and neural origin respectively. Malignant cells retain their tissue-specific IFP, which makes it possible to use these antibodies in tumour diagnosis. Carcinomas are exclusively detected by antibodies to keratin. Monoclonal antibodies to keratin have allowed the differentiation between subgroups of epithelial tumours until now between adenocarcinomas and squamous cell carcinomas. Lymphomas, melanomas and several soft tissue tumours are distinctly recognized by antibodies to vimentin. On the other hand, rhabdomyosarcomas and leiomyosarcomas are positive for desmin, while astrocytomas give a strong reaction with GFAP antibodies. Thus, antibodies to IFP are useful tools for differential diagnosis in surgical pathology.

Lamins are nuclear intermediate filaments that, together with lamin-associated proteins, maintain nuclear shape and provide a structural support for chromosomes and replicating DNA. We have determined the solution structure of the human lamin A/C C-terminal globular domain which contains specific mutations causing four different heritable diseases. This domain encompasses residues 430-545 and adopts an Ig-like fold of type s. We have also characterized by NMR and circular dichroism the structure and thermostability of three mutants, R453W and R482W/Q, corresponding to "hot spots" causing Emery-Dreifuss muscular dystrophy and Dunnigan-type lipodystrophy, respectively. Our structure determination and mutant analyses clearly show that the consequences of the mutations causing muscle-specific diseases or lipodystrophy are different at the molecular level.

BACKGROUND

Dynamic remodeling of the actin cytoskeleton requires rapid turnover of actin filaments, which is regulated in part by the actin filament severing/depolymerization factor cofilin/ADF. Two factors that cooperate with cofilin are Srv2/CAP and Aip1. Human CAP enhances cofilin-mediated actin turnover in vitro, but its biophysical properties have not been defined, and there has been no in vivo evidence reported for its role in turnover. Xenopus Aip1 forms a cofilin-dependent cap at filament barbed ends. It has been unclear how these diverse activities are coordinated in vivo.

RESULTS

Purified native yeast Srv2/CAP forms a high molecular weight structure comprised solely of actin and Srv2. The complex is linked to actin filaments via the SH3 domain of Abp1. Srv2 complex catalytically accelerates cofilin-dependent actin turnover by releasing cofilin from ADP-actin monomers and enhances the ability of profilin to stimulate nucleotide exchange on ADP-actin. Yeast Aip1 forms a cofilin-dependent filament barbed end cap, disrupted by the cof1-19 mutant. Genetic analyses show that specific combinations of activities mediated by cofilin, Srv2, Aip1, and capping protein are required in vivo.

CONCLUSIONS

We define two genetically and biochemically separable functions for cofilin in actin turnover. One is formation of an Aip1-cofilin cap at filament barbed ends. The other is cofilin-mediated severing/depolymerization of filaments, accelerated indirectly by Srv2 complex. We show that the Srv2 complex is a large multimeric structure and functions as an intermediate in actin monomer processing, converting cofilin bound ADP-actin monomers to profilin bound ATP-actin monomers and recycling cofilin for new rounds of filament depolymerization.

BACKGROUND

A-type lamins are type V intermediate filament proteins encoded by the gene LMNA. Mutations in LMNA give rise to diverse degenerative diseases related to premature ageing. A-type lamins also influence the activity of the Retinoblastoma protein (pRb) and oncogenes such a beta-catenin. Consequently, it has been speculated that expression of A-type lamins may also influence tumour progression.

RESULTS

An archive of colorectal cancer (CRC) and normal colon tissue was screened for expression of A-type lamins. We used the Cox proportional hazard ratio (HR) method to investigate patient survival. Using CRC cell lines we investigated the effects of lamin A expression on other genes by RT-PCR; on cell growth by FACS analysis; and on invasiveness by cell migration assays and siRNA knockdown of targeted genes. We found that lamin A is expressed in colonic stem cells and that patients with A-type lamin-expressing tumours have significantly worse prognosis than patients with A-type lamin negative tumours (HR = 1.85, p = 0.005). To understand this finding, we established a model system based upon expression of GFP-lamin A in CRC cells. We found that expression of GFP-lamin A in these cells did not affect cell proliferation but did promote greatly increased cell motility and invasiveness. The reason for this increased invasiveness was that expression of lamin A promoted up-regulation of the actin bundling protein T-plastin, leading to down regulation of the cell adhesion molecule E-cadherin.

CONCLUSIONS

Expression of A-type lamins increases the risk of death from CRC because its presence gives rise to increased invasiveness and potentially a more stem cell-like phenotype. This report directly links A-type lamin expression to tumour progression and raises the profile of LMNA from one implicated in multiple but rare genetic conditions to a gene involved in one of the commonest diseases in the Western World.

Intermediate filaments in most types of cultured cells coalign with microtubules. Depolymerization of microtubules results in collapse of vimentin and desmin intermediate filaments to the nucleus where they form a perinuclear cap. Collapse can also be induced by microinjection of antibodies against intermediate filament or microtubule proteins. Thus, two filament systems interact with each other. But the molecules mediating this interaction are unknown. One of the candidates for this role is a microtubule motor kinesin. Recent data showed that kinesin is involved in the plus end-directed movement of the membranous organelles along microtubules such as radial extension of lysosomes in macrophages and centrifugal movement of pigment in melanophores. Here we report that injection of the anti-kinesin antibody into human fibroblasts results in the redistribution of intermediate filaments to a tight perinuclear aggregate but had no effect on the distribution of microtubules. Thus, kinesin is involved not only in organelle movement but also in interaction of the two major cytoskeletal systems, intermediate filaments and microtubules.

The functionality of the mitochondrion is primarily determined by nuclear encoded proteins. The mitochondrial functional requirements of different tissues vary from a significant biosynthetic role (liver) to a primarily energy metabolism-oriented organelle (heart). The purpose of this study was to compare the mitochondrial proteome from four different tissues of the rat, brain, liver, heart, and kidney, to provide insight into the extent of mitochondrial heterogeneity and to further characterize the overall mitochondrial proteome. Mitochondria were isolated, solubilized, digested, and subjected to quantitative liquid chromatography-mass spectroscopy. Of the 16,950 distinct peptides detected, 8,045 proteins were identified. High-confidence identification threshold was reached by 1,162 peptides, which were further analyzed. Of these 1,162 proteins, 1,149 were significantly different in content (P and q values < 0.05) between at least 2 tissues, whereas 13 were not significantly different between any tissues. Confirmation of the mitochondrial origin of proteins was determined from the literature or via NH(2)-terminal mitochondrial localization signals. With these criteria, 382 proteins in the significantly different groups were confirmed to be mitochondrial, and 493 could not be confirmed to be mitochondrial but were not definitively localized elsewhere in the cell. A total of 145 proteins were assigned to the rat mitochondrial proteome for the first time via their NH(2)-terminal mitochondrial localization signals. Among the proteins that were not significantly different between tissues, three were confirmed to be mitochondrial. Most notable of the significantly different proteins were histone family proteins and several structural proteins, including tubulin and intermediate filaments. The mitochondrial proteome from each tissue had very specific characteristics indicative of different functional emphasis. These data confirm the notion that mitochondria are tuned by the nucleus for specific functions in different tissues.

The elevated expression of stress proteins is considered to be a universal response to adverse conditions, representing a potential mechanism of cellular defense against disease and a potential target for novel therapeutics, including gene therapy and chaperone-modulating reagents. Recently, a single mutation in the small heat-shock protein human alphaB-crystallin was linked to desmin-related myopathy, which is characterized by abnormal intracellular aggregates of intermediate filaments in human muscle. New findings demonstrate that the high level of expression of stress proteins can contribute to an autoimmune response and can protect proteins that contribute to disease processes.

Hepatic stellate cells are considered to be liver-specific pericytes that play a key role in liver fibrosis. Because these cells express desmin and smooth muscle alpha-actin, they were assumed to be of myogenic origin. This hypothesis became doubtful when it was reported that stellate cells also express glial fibrillary acidic protein and neural cell adhesion molecule. In the present study, we show that activated stellate cells express nestin, a class VI intermediate filament protein originally identified as a marker for neural stem cells. Expression of nestin was first studied during spontaneous activation of stellate cells in culture. Immunohistochemistry showed that nestin-positive stellate cells already appeared at day 3, and nearly all the cells became positive for nestin at day 6 and 15. The immunoreaction was present in filaments except in dividing cells. The presence of messenger RNA transcript for nestin was shown by reverse transcription polymerase chain reaction and sequencing of amplified complementary DNA. We then compared the presence of nestin with that of other intermediate filament proteins and smooth muscle alpha-actin. Immunoblotting showed that the relative concentrations of nestin, desmin, and vimentin increased between day 2 and 6 in primary culture. After the initial increase vimentin leveled off, while nestin and desmin showed a tendency to decrease. This pattern was quite different from that of glial fibrillary acidic protein, which kept declining, and smooth muscle alpha-actin, which increased continuously up to day 13 in culture. We then studied the presence of nestin in normal and CCl4-injured rat liver. In normal liver, minimal immunoreaction for nestin was observed within the liver parenchyma. During induction of fibrosis by carbon tetrachloride, nestin-positive stellate cells appeared at 6 weeks, which was late in comparison with the induction of desmin and smooth muscle alpha-actin. We conclude that nestin is induced in stellate cells during transition from the quiescent to the activated phenotype; culture activation is a stronger stimulus than in vivo activation by injection of CCl4. Taken together with reports on expression of glial fibrillary acidic protein and neural cell adhesion molecule by stellate cells, new experimental studies on the embryonic origin of these cells are required.

The RAD50 gene of Saccharomyces cerevisiae is required for chromosome synapsis and recombination during meiosis and for repair of DNA damage during vegetative growth. The precise role of the RAD50 gene product in these processes is not known. Most rad50 mutant phenotypes can be explained by the proposal that the RAD50 gene product is involved in the search for homology between interacting DNA molecules or chromosomes, but there is no direct evidence for this model. We present here the nucleotide sequence of the RAD50 locus and an analysis of the predicted 153-kD RAD50 protein. The amino terminal region of the predicted protein contains residues suggestive of a purine nucleotide binding domain, most likely for adenine. The remaining 1170 amino acids consist of two 250 amino acid segments of heptad repeat sequence separated by 320 amino acids, plus a short hydrophobic carboxy-terminal tail. Heptad repeats occur in proteins such as myosin and intermediate filaments that form alpha-helical coiled coils. One of the two heptad regions in RAD50 shows similarity to the S-2 domain of rabbit myosin beyond that expected for two random coiled coil proteins.

OBJECTIVE

Squamous cell carcinoma of the head and neck (HNSCC), the sixth most prevalent cancer among men worldwide, is associated with poor prognosis, which has improved only marginally over the past three decades. A proteomic analysis of HNSCC lesions may help identify novel molecular targets for the early detection, prevention, and treatment of HNSCC.

METHODS

Laser capture microdissection was combined with recently developed techniques for protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues and a novel proteomics platform. Approximately 20,000 cells procured from FFPE tissue sections of normal oral epithelium and well, moderately, and poorly differentiated HNSCC were processed for mass spectrometry and bioinformatic analysis.

RESULTS

A large number of proteins expressed in normal oral epithelium and HNSCC, including cytokeratins, intermediate filaments, differentiation markers, and proteins involved in stem cell maintenance, signal transduction, migration, cell cycle regulation, growth and angiogenesis, matrix degradation, and proteins with tumor suppressive and oncogenic potential, were readily detected. Of interest, the relative expression of many of these molecules followed a distinct pattern in normal squamous epithelia and well, moderately, and poorly differentiated HNSCC tumor tissues. Representative proteins were further validated using immunohistochemical studies in HNSCC tissue sections and tissue microarrays.

CONCLUSIONS

The ability to combine laser capture microdissection and in-depth proteomic analysis of FFPE tissues provided a wealth of information regarding the nature of the proteins expressed in normal squamous epithelium and during HNSCC progression, which may allow the development of novel biomarkers of diagnostic and prognostic value and the identification of novel targets for therapeutic intervention in HNSCC.

Desmosomes are highly organized intercellular junctions that provide mechanical integrity to tissues by anchoring intermediate filaments to sites of strong adhesion. Transcriptional regulation of desmosomal cadherins specifies their expression pattern and assembly into junctions of distinct composition, thus tailoring desmosome functions in adhesion and morphogenesis within different cells and complex tissues. Desmosome assembly and disassembly are regulated post-translationally by calcium, kinase/phosphatase activity, proteolytic processing, and cross talk with adherens junctions. Post-translational events also govern the level of non-junctional forms of plakoglobin and plakophilins. These armadillo proteins participate in various nuclear functions, in some cases transducing signals regulating cell growth and differentiation.