Step contrast reversal in LEEM during Pb deposition on W(110).

Anomalous step contrast in low energy electron microscopy (LEEM) images observed during Pb deposition on a W(110) surface is discussed. The steps are dark on the clean surface, and become bright by Pb deposition at about 200 °C. The contrast reversal is related to the presence of a two-dimensional (2D) Pb gas on the surface and its atomic density distribution. Upon further deposition the steps become dark again and show an anomalous intensity profile. This change is attributed to the 2D crystallization process.

Characterization of spectroscopic photoemission and low energy electron microscope using multipolarized soft x rays at BL17SU/SPring-8.

Spectroscopic photoemission and low energy electron microscope (SPELEEM) improved its performance after installation at BL17SU/SPring-8, where a multipolarization-mode undulator is employed to produce circularly and linearly polarized soft x rays. This undulator enables us to study the domain structures of ferromagnetic and antiferromagnetic materials by x-ray magnetic circular dichroism and x-ray magnetic linear dichroism. SPELEEM is used to study light elements (C, N, and O), 3d transition-metal elements and 4f rare earth elements, utilizing a wide range of photon energies. The two cylindrical mirrors adopted in front of SPELEEM ensure an illumination area of 14 x 14 microm(2) on the samples. The lateral resolution of a secondary electron photoemission electron microscope image is estimated to be better than 85 nm, whereas the energy resolution of the instrument is better than 0.4 eV.

In vivo occurrence of p16 (MTS1) and p15 (MTS2) alterations preferentially in non-small cell lung cancers.

Frequent homozygous deletions of the p16 (MTS1) gene encoding a cyclin-dependent kinase inhibitor were recently reported in various tumor cell lines including examples derived from lung cancers, but direct evidence for their occurrence in lung cancer patients has not been reported thus far. In the present study, alterations of p16 and/or p15, a p16-related cyclin-dependent kinase, were observed not only in lung cancer cell lines but also in the corresponding tumor specimens in vivo, excluding the possibility of in vitro artifacts. Interestingly, a clear specificity was also noted in terms of the affected histological subtype; i.e., only non-small cell lung cancers carried alterations (6 of 20 as compared to 0 of 20 small cell lung cancer cell lines).

p27KIP1 in human lung cancers: differential changes in small cell and non-small cell carcinomas.

Small cell lung cancers (SCLCs) and non-small cell lung cancers (NSCLCs), two major categories of human lung cancers, have been shown to exhibit considerably different clinicopathological, biological, and molecular genetic characteristics. Inactivation of cyclin-dependent kinase inhibitors is now thought to play an important part in the pathogenesis of this fatal disease. In the present study, we show that in vitro p27KIP1 expression was associated with cell density-dependent growth inhibition in human lung epithelial cells in vitro, whereas in vivo, p27KIP1 expression in lung cells showed an inverse correlation with proliferative activity in the developing and adult normal lungs. Our immunohistochemical examination of 166 lung tumor specimens also revealed a striking difference in p27KIP1 expression between SCLCs and NSCLCs. Of 149 NSCLCs, 107 (72%) showed reduced p27KIP1 expression, with 8 being virtually negative. Furthermore, p27KIP1 expression status was found to be a significant prognostic factor for patient survival in the analysis of the 149 primary, resected NSCLC cases (P = 0.03 by the log-rank test). In contrast, all SCLC specimens thus far examined exhibited significantly increased staining when compared to the corresponding normal lung epithelium. These findings provide additional evidence for the heterogeneity prevalent in human lung cancers and suggest that p27KIP1 might play distinct biological roles in the pathogenesis of the two major histological categories, warranting additional studies to elucidate the functional consequences of such differences.

Predominantly tumor-limited expression of a mutant allele in a Japanese family carrying a germline p53 mutation.

Germline mutations of p53 have been implicated as a cause of cancer susceptibility in the Li-Fraumeni syndrome. Since inactivation of p53 has been suggested to play an important causative role in lung cancer, the present study of the prevalence of germline mutations in 148 patients with this neoplasm was performed. None of 138 randomly chosen patients were found to carry such mutations, while a single patient had a nonsense mutation at codon 213 among 10 patients selected for early onset and/or occurrence of multiple primary cancers. In contrast to the previous report of biallelic expression of p53 in a case with a germline missense mutation, preferential expression of the wild-type allele was observed in the heterozygous state in both normal lung and peripheral blood lymphocytes of our case, whereas expression of mutant mRNA was readily detectable in her lung cancer in the absence of the remaining wild-type allele. Interestingly, the family history of the proband showed a mild aggregation of adulthood cancers and a high prevalence of stomach cancer, a rare component in American families affected by the syndrome. These observations suggest the presence of heterogeneity with regard to molecular and clinical features of germline p53 mutations.

Search for in vivo somatic mutations in the mitotic checkpoint gene, hMAD1, in human lung cancers.

We previously reported the presence of mitotic check-point impairment in about 40% of lung cancer cell lines. To gain an insight into the molecular basis of this impairment, we examined 49 lung cancer specimens for alterations in the hMAD1 mitotic checkpoint gene and identified a somatic, non-conservative missense mutation, which substitutes alanine (GCG) for threonine (ACG) at codon 299, together with a number of amino acid substituting, single nucleotide polymorphisms. This is the first demonstration of hMAD1 mutation in any type of human cancers. The present finding marks hMAD1 as a potential target, although with low frequency, for genetic alterations in lung cancer. Thus, further studies of hMAD1 dysfunction caused by other mechanisms appear to be warranted, as well as potential involvement of other components of the mitotic checkpoint.

Allelic-expression imbalance of the insulin-like growth factor 2 gene in hepatocellular carcinoma and underlying disease.

It has been well documented that the liver is an exceptional organ in which the monoallelic expression of insulin-like growth factor 2 (IGF2) due to genomic imprinting is relaxed during the postnatal period, resulting in biallelic expression thereafter. In the present study, changes in the status of genomic imprinting were examined in 15 hepatocellular carcinomas (HCCs) as well as in 29 liver biopsies of chronic hepatitis or liver cirrhosis without clinical evidence of HCC, following screening for heterozygotes with an ApaI polymorphism in IGF2 in 34 HCCs and 80 such non-HCC cases. Extreme allelic-expression imbalance, leading to restoration of monoallelic IGF2 expression, was observed in 15 (100%) of 15 informative HCCs for the polymorphism with this monoallelic IGF2 expression appearing to be non-random from the paternal allele. Interestingly, the same allelic-expression imbalance was also present in a significant fraction of noncancerous liver specimens of patients with underlying disease known to be associated with HCC development. In contrast, the status of genomic imprinting of H19, another gene closely mapped at 11p15 under opposite imprinting, was strictly maintained in seven (100%) of seven cases informative for an RsaI polymorphism of H19. Together with the previous reports on altered genomic imprinting of IGF2 and H19 in embryonal lesions such as Wilms tumors as well as in lung cancers, the results suggest that perturbations of imprinting status occur as locus and tumor-type specific events in the development of human cancers.

Prognostic significance of abnormal p53 accumulation in primary, resected non-small-cell lung cancers.

PURPOSE: This study was conducted to evaluate the prognostic significance of p53 abnormalities in primary, resected non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Methodologic validation of immunohistologic detection of p53 abnormalities in routine pathology sections was assessed using 31 lung cancer specimens for which p53 gene status was known from our previous molecular biologic studies. Applying the optimized cutoff value, we evaluated the prognostic significance of p53 abnormalities in an independent cohort of 208 NSCLC patients with complete follow-up data, whose resections were consecutively performed between January 1984 and December 1988. RESULTS: Immunohistologic detection of p53 abnormalities appeared to be reliable and showed approximately 90% concordance with the p53 gene status. Using the selected cutoff value of 10%, 46% of 208 NSCLCs showed p53 abnormalities. There was no relationship between p53 abnormalities and clinical outcome in the entire cohort, which represented all histologic subtypes of NSCLC (P = .58). Based on the reasoning that the influence of p53 abnormalities may have been obscured by distinct biologic roles depending on histologic subtypes, we also separately analyzed subsets of patients with adenocarcinomas (n = 100) and with squamous cell carcinomas (n = 88) and found that it may be a useful prognosticator only in adenocarcinoma patients (P = .04). CONCLUSION: p53 abnormalities are not a significant prognostic factor in primary, resected NSCLC when all histologic subtypes are combined, but may be a useful prognosticator for adenocarcinomas. Additional studies are warranted for further evaluation, specifically of adenocarcinomas.

High frequency of clonally related tumors in cases of multiple synchronous lung cancers as revealed by molecular diagnosis.

In patients with multiple synchronous lung tumors, discrimination of multicentric lung cancers from intrapulmonary metastasis is important for treatment decision, but this is sometimes difficult. The aim of this study was to retrospectively distinguish multicentric lung cancers from intrapulmonary metastases in 14 such cases by loss of heterozygosity (LOH) and p53 mutational status. DNA was extracted from microdissected tumor cells in paraffin-embedded archival tissue, and 3p14.2, 3p21, 3p25, 9p21, and 18q21.1 were investigated for LOH. Exons 5-8 of the p53 gene were examined for mutations by the PCR, followed by single-strand conformation polymorphism analysis and DNA sequencing. For cases with the same LOH pattern, we calculated a clonality index, the probability of the given LOH pattern when these tumors were hypothesized to be independent in origin. Eleven of 14 cases (79%) were thus diagnosed as having pulmonary metastasis and only one case as having genuinely multicentric lung cancers. Two cases presented difficulty in diagnosis. In several cases, the LOH patterns conflicted with p53 mutation patterns, suggesting that clonal evolution is directly affected by certain genetic changes. The combination of p53 with LOH helped increase both the sensitivity and specificity of the assay.

Prognostic significance of cyclin D1 and retinoblastoma expression in combination with p53 abnormalities in primary, resected non-small cell lung cancers.

This study was conducted to evaluate the prognostic significance of cyclin D1 and retinoblastoma (Rb) expression in combination with abnormal p53 accumulation in primary, resected non-small cell lung cancers (NSCLCs). We evaluated immunohistochemically the expression of cyclin D1 and Rb in 208 NSCLC patients whose resections were consecutively performed between January 1984 and December 1988 and determined their prognostic significance by comparison with follow-up data. Expression of cyclin D1 and Rb was detected immunohistochemically in 39 and 80% of the 208 NSCLCs, respectively. The Kaplan-Meier survival curve demonstrated that absence of cyclin D1 expression was significantly associated with shortened survival (P = 0.01 by the log-rank test), particularly in adenocarcinomas (n = 100; P = 0.004). Expression status of the Rb protein was not significantly associated with clinical outcome in any of the cohorts. The predictive power of these prognosticators was also assessed in combination with findings for abnormal p53 accumulation by multivariate analysis using the Cox proportional hazards modeling. Patients with cyclin D1-negative tumors had a significantly greater risk of earlier death than those with cyclin D1-positive tumors (risk ratio, 1.61; P = 0.03), particularly in adenocarcinomas (risk ratio, 2.49; P = 0.002). Cases showing abnormal p53 accumulation tended to have a shortened survival only when the tumors were adenocarcinomas (risk ratio, 1.75; P = 0.06). Absence of cyclin D1 expression may be a useful prognosticator for shortened survival in primary, resected NSCLCs with particular significance for adenocarcinomas. In contrast, Rb expression status is not a useful prognostic factor for any type of NSCLC.

Gains of 1q21-q22 and 13q12-q14 are potential indicators for resistance to cisplatin-based chemotherapy in ovarian cancer patients.

The mechanism of drug resistance in ovarian cancer is multifactorial, and accumulation of multiple genetic changes may lead to the drug-resistant phenotype. In our attempt to find characteristic genetic changes in drug-resistant tumors, we screened the whole genome for gene aberrations in 28 primary ovarian cancers using the comparative genomic hybridization method. These cancers included 14 tumors from patients who did not respond to cisplatin-based combination chemotherapy and 14 tumors from patients who had complete response to the chemotherapy. We found gains in chromosomal regions 1q21-q22 and 13q12-q14 to be related to the drug-resistant phenotype in ovarian cancer patients. Several genes encoding transcription factors, oncogenes, cell cycle regulators, and regulators of the apoptotic pathway are located on these regions of the chromosomes, and these genes are potential modulators for toxic insults in cancer cells. This is the first report that shows the relationship between certain genomic aberrations and clinical resistance to cisplatin-based chemotherapy in ovarian cancer patients based on the comparative genomic hybridization analysis. Present findings suggest that these chromosomal gains may be potential indicators for prediction of resistance in ovarian cancer patients before cisplatin-based chemotherapy.

Cloning and expression of the leucine gene from Thermus thermophilus in Escherichia coli.

A pBR322-T. leu hybrid plasmid was constructed which contains a 3.75 Md HindIII-fragment derived from Thermus thermophilus HB27 chromosomal DNA. In the Escherichia coli host, this plasmid coded for the beta-IPM dehydrogenase (product of leuB) activity, the optimal temperature of which was 80 degrees C, suggesting that information on the thermostability of the enzyme lies in its structural gene. 10-day propagation of E. coli [pBR322-T.leu] at 37 degrees C decreased the temperature optimum from 80 degrees to 75 degrees C. This change, which was found to depend on the plasmid but not on the host cells, might be due to selection of some mutation at the non-restrictive temperature of 37 degrees C. Our results suggest that the 3.75 Md HindIII-fragment of pBR322-T.leu carries a promoter of the thermophile, which could function in E. coli.

Secretion of a new growth factor, smooth muscle cell derived growth factor, distinct from platelet derived growth factor by cultured rabbit aortic smooth muscle cells.

In attempts to determine the mechanism of proliferation of arterial smooth muscle cells (SMC) in intimal atheromatous lesions, autocrine secretion of growth factors by SMC has recently received much attention. Here we report a new growth factor named smooth muscle cell derived growth factor (SDGF). Cultured rabbit medial SMC secreted SDGF for 1 week during their incubation in serum-free media only after at least 4 passages. SDGF differed from platelet derived growth factor (PDGF) physicochemically, immunologically, and biologically. The properties of SDGF also seemed different from those of other known growth factors that stimulate the proliferation of mesenchymal cells.

Secretion of a potent new migration factor for smooth muscle cells (SMC) by cultured SMC.

Migration of smooth muscle cells (SMC) in the arterial wall is important in the formation of intimal thickening. In this work, cultured SMC from the rat and rabbit aortic media at 2nd to 12th passages were found to secrete a potent migration factor for SMC which was named SMC-derived migration factor (SDMF). This factor stimulated the migration of SMC dose-dependently and its maximum activity was 2-8 times that of PDGF. Checker board analysis showed that SDMF was chemotactic, but not chemokinetic. In further studies, SDMF was found to be inactivated at 100 degrees C for 10 min or by trypsinization, but not inactivated by mercaptoethanol. This factor was not dialyzable. Molecular weight was approximately 500 kDa by a gel filtration. The activity was not inhibited by an anti-PDGF antibody or a fibronectin antiserum. These data suggest that SDMF is a potent migration factor for SMC and that SDMF is distinct from PDGF, fibronectin or other known migration factors. This autocrine system of secretion of SDMF by SMC and its induction of SMC migration may contribute to intimal thickening of the arterial wall in atherosclerosis.

Effects of transforming growth factor-beta 1 on growth of aortic smooth muscle cells. Influences of interaction with growth factors, cell state, cell phenotype, and cell cycle.

Transforming growth factor (TGF)-beta 1 may have different effects on cell proliferation depending on many conditions. This paper clarifies the effects of various conditions on the effect of TGF-beta 1 on proliferation of cultured rabbit aortic smooth muscle cells (SMC) and also the time of its action during the cell cycle. TGF-beta 1 at 10-10,000 pg/ml inhibited DNA synthesis of SMC in the G0 stage derived from normal media or atheromatous intima stimulated by either platelet-derived growth factor (PDGF), fibroblast growth factor, SMC-derived growth factor, or fetal bovine serum (FBS). TGF-beta 1 also inhibited the growth of SMC in the growing state stimulated by either PDGF or FBS. TGF-beta 1 was effective only when added to the culture within 2 h after stimulation of the G0 state SMC with PDGF. It also inhibited increase in transcription of the c-myc protooncogene on stimulation of SMC with PDGF. These data suggest that TGF-beta 1 inhibited proliferation of SMC irrespective of the cell phenotype, growth conditions, and growth factors present and that it exerted this inhibitory effect during the time of the G0/G1 transition.

Effects of smooth muscle cell derived growth factor (SDGF) in combination with other growth factors on smooth muscle cells.

Recently intimal thickening was shown to be due to stimulation of proliferation of arterial smooth muscle cells (SMC) by autocrine secretion of growth factor(s). We have reported that cultured rabbit aortic SMC secrete a growth factor (SDGF) distinct from other known growth factors. This paper reports on studies on the biological characteristics of SDGF. Putative "competence" and "progression" growth factors synergistically stimulated DNA synthesis of cultured rabbit aortic SMC. Conditioned medium (CM) from SMC containing SDGF stimulated DNA synthesis in SMC synergistically with either the competence factors or the progression factors. This synergistic effect was also observed in the presence of optimal concentrations of both competence and progression factors. The continuous presence of CM was essential for its stimulation of DNA synthesis whereas the presence of PDGF for only the first 4 h of culture was sufficient to induce maximum stimulation of DNA synthesis. These results suggest that SDGF is a new growth factor distinct from either competence or progression factors and that it stimulates a different pathway in SMC from those stimulated by other known growth factors.

Clinicopathologic study of CD56 (NCAM)-positive angiocentric lymphoma occurring in sites other than the upper and lower respiratory tract.

The expression of the neural cell adhesion molecule (NCAM) (CD56, NKH-1) is a rare phenomenon in malignant lymphoma. Recently, several authors, including our group, described the clinicopathologic, phenotypic, and genotypic features of NCAM-positive tumors as a unique subgroup within a larger category of hematolymphoid malignancies. Ten cases of CD56+ angiocentric lymphoma occurring in sites other than the upper aerodigestive tract were studied for evaluating their characteristics. The disease occurred in six men and four women varying from 24 to 85 years (mean age, 53 years) who often exhibited a striking predilection for extranodal sites of involvement, such as the skin, gastrointestinal tract, and muscle, usually in the absence of peripheral lymphadenopathy. Although the cytologic appearances and immunophenotypic profile varied from case to case, these tumors often exhibited azurophilic granules, an angiocentric growth pattern, and surface CD3-, T-cell receptor (TCR) antigens-, and CD56+ phenotype without B-cell phenotype, except for a single case of CD3+, TCR alpha/beta+, and CD56+ phenotype. Genotype investigation exhibited germline configuration of the TCR beta and gamma chain genes and the immunoglobulin heavy chain gene in all five cases of surface CD3- phenotype examined, whereas the case of CD3+ phenotype showed rearrangement of TCR beta. They seem to constitute a distinct entity of the lineage spectrum spanning from natural killer (NK) cell to NK-like T cell.

Clinicopathologic study of PRAD1/cyclin D1 overexpressing lymphoma with special reference to mantle cell lymphoma. A distinct molecular pathologic entity.

Mantle cell lymphomas (MCLs) are frequently associated with the overexpression of PRAD1/cyclin D1, activated by 11q13 translocation and its molecular counterpart BCL-1 gene rearrangement. We recently described the correlation of positive nuclear staining using monoclonal antibody against a PRAD1/cyclin D1 product with mRNA overexpression in MCLs. In the present study, we immunohistochemically investigated the PRAD1/cyclin D1 protein in a large series of 334 lymphoproliferative disorders, including 39 cases of MCLs on paraffin sections. Based on the cyclin D1 positivity, CD5 expression, and the morphologic features of the tumor tissue, four groups of MCL-related lesions were identified among the B-cell lymphomas examined: 36 cases with cyclin D1 overexpression, 35 (95%) of which exhibited CD5-positivity and MCL-morphology (Group 1); four cases of lymphomas with MCL morphology and CD5 expression but lacking cyclin D1 overexpression (Group II); four cases of lymphomas without cyclin D1 overexpression and surface CD5 but that fall within the morphologic boundaries of MCLs (Group III); and 11 cases of CD5-positive diffuse large cell lymphomas without cyclin D1 overexpression (Group IV). The Group I cases demonstrated quite homogeneous clinicopathologic features identical to those of MCLs. This group showed a poor prognosis (11% had 5-year survival), which is highly contrasted with that of Group II (100%). Although the four groups of MCL-related lesions sometimes overlapped in their histologic or phenotypic spectrums, each appeared to show distinct clinicopathologic and prognostic profiles. Our study provides a basis for further clarification of the nature of the neoplasms of Groups II, III, and IV. Moreover, this comprehensive study may indicate that the overexpression of PRAD1/cyclin D1 is biologically essential to defining MCLs.

Anaplastic large cell lymphoma: a distinct molecular pathologic entity: a reappraisal with special reference to p80(NPM/ALK) expression.

The p80(NPM/ALK) expression activated by the t(2;5) (p23;q35) translocation recently has been shown to play an important role in the pathogenesis of anaplastic large cell lymphoma (ALCL). However, the clinicopathologic significance of identification of p80 among ALCL cases has not been completely resolved. Difficulties also exist in the histologic and immunophenotypic identification of ALCL and Hodgkin's disease (HD) as separate processes, often complicating the clinicopathologic evaluation of and therapeutic approach to these entities. In order to clarify these issues, 67 specimens of ALCL and 63 specimens of HD (31 of the nodular-sclerosing type [NS-HD] and 32 of the mixed-cellularity type [MC-HD]) were immunostained using anti-p80 antibody and other relevant markers on paraffin sections. The clinicopathologic and immunophenotypic features were reviewed on the basis of p80 reactivity. The expression of p80 was detected in 43 of 67 cases of ALCL (64%), but none of HD. The p80+ ALCL cases constituted a very homogeneous group of tumors, characterized by the occurrence in a much younger group and relatively more favorable clinical course than the p80- ALCL, which were in keeping with the data previously reported. They showed virtually the identical immunophenotypic findings of p80+, CD30+, EMA+, CD15-, bcl-2-, and Epstein-Barr virus (EBV) with T- and null-cell phenotype, and showed the distinct morphologic features, including three cases of lymphohistiocytic/small-cell variant, as follows: the indented nuclei, often termed as reniform, embryolike, and horseshoelike; multiple, irregular, but indistinct nucleoli; and few reactive cells of eosinophils and epithelioid cells. Conversely, the 24 p80- ALCL cases, in which epithelial membrane antigen (EMA) and bcl-2 positivities were 33% and 55%, respectively, were heterogeneous and could be subdivided into five different categories, namely (a) 11 cases of HD-like ALCLs, (b) six cases of p80 common ALCL, (c) three cases of secondary ALCL, (d) two cases of primary cutaneous ALCL, and (e) two cases of primary classical ALCL that lacked p80 expression. This study clearly demonstrated that the immunohistochemical detection of p80 is of a crucial importance in delineating the biologically distinct entity of "primary classical ALCL" from various diseases that show morphologic and immunohistologic overlap, including HD and HD-like ALCL.