Quantification of immunohistochemistry--issues concerning methods, utility and semiquantitative assessment II.

Immunohistochemistry is entering its fourth decade of use on formalin-fixed paraffin-embedded tissues. Over this period the method has evolved to become a major part of the practice of diagnostic surgical pathology worldwide. From the beginning immunohistochemistry has been adapted to provide a range of markers of cell lineage and tissue type, with particular application to the diagnosis and classification of tumours. In this modality immunohistochemical methods were employed simply as 'special stains', the results of which were evaluated qualitatively by the pathologist, as for any other stain. More recently, attention has shifted to the demonstration of prognostic markers in tumour cells, driven by the advent of molecular biology and the discovery of numerous regulatory molecules, coupled with manufacture of the corresponding specific antibodies. Immunohistochemistry has rapidly adapted to this new use, but in so doing the demand for quantification has become paramount; it is no longer enough that the 'stain' is there; rather it is a question of 'How much is there?'. This review explores the limitations of immunohistochemistry when employed in a semiquantitative mode, and explores the possibility of fulfilling the full potential of immunohistochemistry, as a true quantitative immunoassay applied in a tissue section environment.

Dynamic in vivo interactions among Myc network members.

Members of the Myc oncoprotein network (c-Myc, Max, and Mad) play important roles in proliferation, differentiation, and apoptosis. We expressed chimeric green fluorescent protein (GFP) fusions of c-Myc, Max, and three Mad proteins in fibroblasts. Individually, c-Myc and Mad proteins localized in subnuclear speckles, whereas Max assumed a homogeneous nuclear pattern. These distributions were co-dominant and dynamic, however, as each protein assumed the pattern of its heterodimeric partner when the latter was co-expressed at a higher level. Deletion mapping of two Mad members, Mad1 and Mxi1, demonstrated that the domains responsible for nuclear localization and speckling are separable. A non-speckling Mxi1 mutant was also less effective as a transcriptional repressor than wild-type Mxi1. c-Myc nuclear speckles were distinct from SC-35 domains involved in mRNA processing. However, in the presence of co-expressed Max, c-Myc, but not Mad, co-localized to a subset of SC-35 loci. These results show that Myc network proteins comprise dynamic subnuclear structures and behave co-dominantly when co-expressed with their normal heterodimerization partners. In addition, c-Myc-Max heterodimers, but not Max-Mad heterodimers, localize to foci actively engaged in pre-mRNA transcription/processing. These findings suggest novel means by which Myc network members promote transcriptional activation or repression.

Non-invasive image acquisition and advanced processing in optical bioimaging.

Light is a most versatile tool for investigating biological systems and phenomena; the range, non-destructiveness, spatial discrimination and speed of optical imaging are all important for investigating structure and function at the cellular, tissue or even whole organism level. In live biological imaging, where the technological requirements are heightened, other features of light, such as coherence and wavelength, are used to generate the additional contrast and resolution needed. We report here recent improvements in our ability to image biological specimens optically, focusing on (a) spectral resolution and the related image processing issues, and (b) tomographic three-dimensional fluorescence imaging in vivo.

Identification of focal viral infections by confocal microscopy for subsequent ultrastructural analysis.

A correlative microscopy method for the ultrastructural analysis of focal viral tissue infections is presented. Using a confocal scanning laser microscope, foci of infection are identified in tissue sections prior to embedment; a variety of techniques can be employed for viral detection, including staining with standard histochemical reagents and fluorescently labeled antibodies. Areas of infection identified using confocal microscopy are excised from the tissue sections, embedded, and examined by transmission electron microscopy. Applications of this technique in both diagnostic and basic research settings are described.

Glucocorticoid modulation of transformed cell proliferation is oncogene specific and correlates with effects on c-myc levels.

In the presence of the glucocorticoid hormone dexamethasone, bovine papillomavirus-1 (BPV-1)-transformed C127 mouse fibroblasts assume a flattened morphology and reach a saturation density of only 50% of that attained without hormone. This phenotypic reversion of transformation is dependent on the continued presence of dexamethasone and occurs with concentrations as low as 1 nM. Dexamethasone also suppresses the growth of the parental C127 cells as well as that of cells transformed by polyoma middle-T. In contrast, the growth of C127 cells transformed by the oncogenes v-H-ras, v-mos, or v-fes is inhibited by low concentrations of dexamethasone (1 nM) and stimulated by higher concentrations (0.1-1 microM), possibly due to dexamethasone-induced transcription from the viral long terminal repeat promoters as is shown for v-H-ras. On the other hand, inhibition of BPV-transformed cell line growth by dexamethasone does not appear to be related to hormone effects on BPV-1 oncogene transcription. Indeed, in several cases, dexamethasone increases the steady state transcript levels of the BPV-1 oncogenes, E5 and E6-E7, while suppressing cellular proliferation. Dexamethasone also rapidly reduces the steady state levels of c-myc in the BPV-transformed cells but has less effect on c-myc expression in the ras-transformed cells. These results demonstrate that the growth-promoting actions of the papillomavirus transforming genes, but not those of several retroviral oncogenes, may be overcome by dexamethasone, which appears to act by down-regulation of c-myc expression.

Giant two-dimensional gel electrophoresis: methodological update and comparison with intermediate-format gel systems.

Two-dimensional (2-D) gel electrophoresis methods for separating complex mixtures of proteins have not changed fundamentally since their original description in the late 1970's. Nevertheless, 2-D gel resolution has improved substantially as a result of a series of incremental modifications. One of these was the development of the "giant-gel" format, using gels measuring at least 30 x 30 cm to provide the highest resolution 2-D gel system available. As originally described, this procedure has several important limitations: it requires custom-built equipment; it is expensive in terms of time, reagents, film and support matrices; and it generates gels which are difficult to manupulate, particularly for silver staining. This report describes modifications in the giant gel procedure to permit use of a commercially available gel apparatus and to obtain giant gels of improved mechanical strength suitable for silver staining. The resolution of giant gels is compared with that obtained using two systems currently being marketed for use by laboratories performing large numbers of 2-D gel analyses. The smaller format gels resolved fewer proteins, by 30-40%, compared with the giant gels. This difference in resolving power suggests that giant gels will continue to be useful in selected applications.

Insulin-stimulated protein tyrosine phosphorylation in intact cells evaluated by giant two-dimensional gel electrophoresis.

We have studied the insulin-stimulated phosphorylation of proteins in NIH 3T3 cells expressing high numbers of human insulin receptors (HIR 3.5 cells) using the technique of giant two-dimensional gel electrophoresis. In serum-deprived cells, insulin stimulated the phosphorylation of more than 25 proteins; all but two of these were also phosphorylated in response to 15% (v/v) fetal bovine serum, which also stimulated the phosphorylation of additional proteins thought to be direct substrates for protein kinase C. In cells pretreated insulin specifically stimulated the phosphorylation insulin specifically stimulated the phosphorylation of at least 26 predominantly cytosolic proteins, only one of which was observed in insulin-treated cells not exposed to phenylarsine oxide. Serum was without effect in cells pretreated with phenylarsine oxide. In phenylarsine oxide-pretreated cells, phosphoamino acid analysis of 10 of the most highly labeled insulin-stimulated phosphoproteins showed that all 10 were labeled predominantly or exclusively on tyrosine residues. The phosphorylation of several of these could be stimulated in vitro by the addition of insulin to a detergent extract of cells in the presence of Mn2+ and ATP. In general, the insulin-stimulated phosphorylations observed in the presence of phenylarsine oxide were more rapid than those observed in its absence. Finally, a variety of other growth factors and mitogens did not stimulate any of the insulin-stimulated phosphorylations in the presence of phenylarsine oxide. Thus, the use of this inhibitor apparently unmasked a number of novel insulin-specific protein phosphorylations that were ordinarily undetectable. We suggest that at least some of these proteins may be direct substrates for the insulin receptor protein tyrosine kinase and may play significant roles in insulin action.

Papillomavirus-associated inductions of cellular proteins in mouse C127 cells: correlation with the presence of open reading frame E2.

Bovine papillomavirus type 1 (BPV-1) readily transforms mouse C127 cells, conferring the ability to grow in soft agar and to form tumors in athymic (nu/nu) mice. Electrophoresis of total cellular proteins from these BPV-transformed lines on ultra-high resolution, giant two-dimensional gels displays the presence of novel, papillomavirus-related protein phenotypes. Analysis of the established BPV-1-transformed C127 cell lines, ID13 and ID14, reveals a set of six proteins which are either absent or synthesized at extremely low levels in the parental cell line. One of these proteins is also present in v-ras-transformed C127 cells, but none of the others are found in cells transformed by a variety of viral oncogenes, including the polyomavirus middle T, v-mos, or v-fes. The genome of BPV-1 contains two separate open reading frames (ORFs), E5 and E6, which can act independently to transform C127 cells. In addition, trans-activator and repressor proteins encoded respectively by the full-length and carboxy-terminal E2 ORF regulate the level of expression of other BPV-1 genes. We examined 34 cell lines transformed by intact and subgenomic recombinant DNAs of BPV-1. Cells harboring BPV-1 DNAs engineered to eliminate the expression of ORFs E4, E5, E6, or E7 display five of the PV-associated proteins, but these proteins are not seen in lines lacking the full E2 ORF. Moreover, G418-selected nontransformed cells expressing E2 cDNA from an SV40 promoter exhibit these proteins at high levels. Surprisingly, these proteins are also present in cells containing BPV-1 DNAs with amino-terminal E2 deletions, suggesting that these PV-associated proteins represent novel cellular responses to a factor encoded within the E2-C gene region.

Insulin rapidly induces the biosynthesis of elongation factor 2.

Insulin increases the rate of overall protein synthesis in many cells and tissues, while inducing the preferential expression of individual proteins. To identify and characterize such proteins, NIH 3T3 cells stably expressing more than 10(6) human insulin receptors per cell (HIR 3.5; Whittaker, J., Okamoto, A. K., Thys, R., Bell, G. I., Steiner, D. F., and Hofmann, C. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5237-5241) were treated with insulin in the presence of [35S]methionine, and labeled proteins were separated using ultra-high resolution ("giant") two-dimensional gel electrophoresis. Overall protein synthesis was enhanced as much as 3-fold by insulin treatment; the synthesis of approximately 1% of the 2,500 proteins visible on the gel autoradiographs was further selectively increased. By using immunoblotting, immunoprecipitation and comigration assays, we identified one of the rapidly induced proteins (Mr 96,000; pI 6.8) as eukaryotic elongation factor 2 (EF-2), a major component of the protein translation apparatus. Insulin induced the synthesis of EF-2 within 20 min of treatment, with a half-maximal dose of 10(-11) M. It was synthesized as a precursor form that was processed to a more basic mature species within 30 min. Long term treatment with insulin led to accumulation of EF-2 within the cell and prevented the substantial decrease in EF-2 concentration that occurred during serum deprivation. Finally, we found that insulin induction of EF-2 occurred normally in the presence of the RNA-transcription inhibitor, actinomycin D. Thus, insulin rapidly induced the synthesis of EF-2 predominantly or exclusively at the level of mRNA translation.

Behavioral approaches to secondary prevention of coronary heart disease.

Over the past 10 years behavioral approaches to the treatment of coronary heart disease (CHD) have become widely recognized as being a significant complement to traditional medical and surgical therapies. The success of approaches to secondary prevention now relate to quality, as well as quantity, of life. A multifaceted program, including dietary management, smoking cessation, physical exercise, modification of type A behavior, and psychological counseling are components of many cardiac rehabilitation programs. Behavioral interventions are effective in reducing traditional risk factors for CHD events, and for improving the quality of life among victims of a disease with significant psychological, as well as physical, consequences. However, the effectiveness of behavioral interventions for prolonging life is less certain and requires more careful evaluation. The mechanisms by which behavioral treatments may influence clinical CHD end points is also in need of further investigation.

Fatal pneumonia in an adult due to respiratory syncytial virus.

Respiratory syncytial virus (RSV) infections in adults are generally mild, and no fatalities in uninstitutionalized adults have been reported. To our knowledge, we document herein the first case of fatal RSV pneumonia in a previously healthy elderly woman living at home, in whom complement fixation titers against RSV rose from less than 1:8 to reach 1:256 at death. Cytoplasmic inclusions characteristic of RSV were detected at autopsy.

Expression of genes for the alpha and beta subunits of the Na,K-ATPase in normal and drug resistant cells.

The studies described in this report show that the genes for sodium, potassium-ATPase exhibit more complexity than was originally expected on the basis of studies of the Na,K-ATPase proteins or their transport activities. First, there are at least three isoforms of the alpha subunit. Second, expression of the beta subunit, although apparently always leading to the same protein product, involves complex variability in the sizes and predominance of different mRNA classes. The biologic basis for this heterogeneity of mRNA transcripts is not known. Third, ouabain resistance in at least one cell line, the human Hela C+ cell line, involves amplification of two independent genetic units, the alpha and beta subunit genes, as well as high levels of expression of the mRNAs. These results serve as strong evidence for the importance of the beta subunit, although no discrete functions have yet been assignable to this protein. It is otherwise difficult to understand why the alpha and beta subunits are both amplified in these cells, and both decline upon withdrawal of the selective agent. On the basis of evidence gathered in our laboratory and that of other groups, we conclude that the genetic basis for sodium transport in eukaryotic tissues is far more complex than previously anticipated. There appears to be a need for specialization of sodium, potassium-ATPases in different tissues and for complex generation of multiple beta-subunit mRNA species. Our molecular genetic approach has also allowed us to demonstrate that there is probably an important role played by the beta-subunit in Na,K-ATPase function.(ABSTRACT TRUNCATED AT 250 WORDS)

Low-cost two-dimensional gel densitometry.

A major obstacle to full utilization of the powerful technique of two-dimensional (2-D) gel electrophoresis is the expense and complexity of quantifying the results. Using an analog-to-digital converter already present in the widely available Commodore 64 or Commodore 128 microcomputer, we have developed a 2-D gel densitometer (GELSCAN) which adds only $20.00 to the cost of the Commodore system (currently around $700.00). The system is designed to work with autoradiograms of 2-D gels. Spots of interest are identified visually and then positioned manually over a light source. A pinhole photoelectric sensor mounted in a hand-held, Plexiglas holder, or "mouse," is briefly rubbed over each spot. Maximum density of the spot is determined and its value is converted to counts per minute via an internal calibration curve which corrects for the nonlinear response of film to radiation. Local spot backgrounds can be subtracted and values can be normalized between gels to adjust for variation in amount of radioactivity applied or in exposure time. Reproducibility is excellent and the technique has some practical as well as theoretical advantages over other more complicated approaches to 2-D gel densitometry. In addition, the GELSCAN system can also be used for scanning individual bands in 1-D gels, quantitation of "dot-blot" autoradiograms and other tasks involving transmission densitometry.

Comparative value of bone scintigraphy and radiography in monitoring tumor response in systemically treated prostatic carcinoma.

Radionuclide bone scans and skeletal radiographs were obtained before and during combination chemotherapy or initial hormonal treatment in 46 patients with disseminated adenocarcinoma of the prostate. The purpose of the study was to determine the usefulness of these two modalities in evaluating tumor response to therapy. Prior to treatment, bone scans were positive in 44 patients (96%). In all but one patient either bone radiographs or bone marrow biopsy revealed evidence of osseous metastases. In 22 patients partial response to therapy was documented by a variety of other staging tests. Eleven of these patients showed concurrent or later improvement on bone scans; one showed improvement on a radiograph. "Flare phenomena" were observed relatively frequently since 23% of the scans and 50% of the radiographs showed worsening at the time of response. Bone scans revealed worsening in 79% of 33 patients with disease progression of extraosseous tumor; radiographs were equally sensitive (82% worsening). It is concluded that bone scans in particular are useful for monitoring tumor status in systemically treated patients with prostate cancer. However, because of the lack of sensitivity for response and paradoxical worsening with tumor regression in some patients, scans are not accurate enough to be employed as the sole test in following these patients.

Phase II trial of cisplatin in small cell carcinoma of the lung.

Eighteen patients with histologically documented small cell carcinoma of the lung who had failed initial combination chemotherapy regimens were treated with single-agent cisplatin in a dose of 100 mg/m2 every 3 weeks, with mannitol and fluid diuresis. Tumor regression was limited to one partial response (response rate, 6%; 95% confidence limits. 1%-27%). Significant toxic effects were gastrointestinal (severe nausea and vomiting in 12 of 14 patients) and hematologic (severe leukopenia in one patient and severe thrombocytopenia in three). The antitumor efficacy of high-dose cisplatin in heavily pretreated patients with small cell carcinoma of the lung appears to be marginal.

Small cell carcinoma presenting as an extrapulmonary neoplasm: sites of origin and response to chemotherapy.

Eight (4%) of 203 consecutive prospectively staged and treated patients with small cell carcinoma (SCC) had no evidence of pulmonary or mediastinal tumor on chest roentgenogram or at fiberoptic bronchoscopy at the time of diagnosis. There were two distinct clinical presentations in these SCC patients with exclusively extrapulmonary tumors. Four had discrete localized extrapulmonary neoplasms, presumably originating in these sites. In the other 4 cases with either regional or widely metastatic disease, no obvious primary tumor could be documented in the lungs or elsewhere. One complete and two partial responses to chemotherapy (duration 6 to greater than 11 mo) occurred in 6 evaluable patients. Two remaining patients were inevaluable for response because they received adjuvant chemotherapy after irradiation or excision of the primary tumor and are free of disease at 15 and 28 months. Results document two clinicopathologic entities of extrapulmonary SCC, more firmly establish that it can be responsive to chemotherapy, and encourage systemic therapy as part of initial treatment planning.

Small cell lung cancer: radionuclide bone scans for assessment of tumor extent and response.

Radionuclide bone scans were performed before and during combination chemotherapy in 119 systematically staged patients with small cell carcinoma of the lung. Before therapy, 49 patients (41%) had positive scans. Scan positivity was significantly associated with the presence of metastatic tumor in the bone marrow, positive skeletal radiographs, and elevated serum alkaline phosphatase levels. Nonosseous distant metastases were significantly more likely to be detected as the number of areas of focal abnormalities on bone scan increased. The survival of patients with documented distant metastases in bone and nonosseous sites was significantly inferior to the survival of patients with limited disease, isolated osseous extensive disease, and extensive disease occurring only in nonbony sites. Of 36 patients with initially abnormal scans and tumor regression documented by other methods, scan findings improved in 24 (67%). In 26 (36%) of 72 scans in patients demonstrating disease progression in extraosseous sites, new areas of increased radionuclide uptake appeared. Improvement or worsening in follow-up scans was associated with nonbony tumor response or progression, respectively, 70% of the time. Serial bone scans provide reasonably accurate staging and prognostic information in patients with small cell lung cancer, although they are probably not sufficiently reliable to be used as the sole parameter in therapeutic decision-making.

Stimulation of amniotic fluid cell growth by cartilage growth factor.

Cartilage growth factor (CGF) stimulates the growth of primary and secondary cultures of human amniotic fluid cells. Over a 14--16-day period there is an approximately 70% enhancement in the number of cells in primary cultures and a 170% increase in secondary cultures. Neither the karyotype or the specific activities of lysosomal enzymes are altered by the presence of CGF in the medium.