Haymaker gene expression in malignant and normal gynecologic tissues.

We previously reported that cell lines established from human carcinomas and leukemias/lymphomas expressed high levels of an intracellular membrane-bound protein, Haymaker, whereas cell lines derived from non-malignant connective tissue cells and lymphoid cells expressed low levels of this gene product. To determine whether these findings reflect neoplastic transformation or, alternatively, tissue specificity, we examined by immunohistochemical and molecular methods the expression of Haymaker in gynecologic organs with and without tumor. A highly specific, affinity-purified rabbit polyclonal antibody against a 25-mer Haymaker peptide was used for immunohistochemical staining and morphometric analysis of 85 tissue specimens. Immunohistochemical studies demonstrate, for the first time, that Haymaker protein is highly expressed in epithelial cells of the endometrium of the normal uterus and to a somewhat lesser extent in the mucosa of the normal vagina and cervix, but is poorly expressed or absent in cells of the connective tissue and smooth muscle strata of these organs (p < 0.005). Significant differences in Haymaker expression, as assessed by immunohistochemistry, between malignant and normal gynecologic tissues were not observed (p = 0.27). The expression of Haymaker protein does not appear, therefore, to be a marker of malignant transformation of the epithelium of gynecologic organs but rather distinguishes both normal and malignant epithelial cells from normal connective tissue and smooth muscle cells.     

Expression of TRAIL and TRAIL receptors in normal and malignant tissues

TRAIL, tumor necrosis factor-related apoptosis-inducing ligand, is a member of the TNF family of proteins.Tumour cells were initially found to have increased sensitivity to TRAIL compared with normal cells, raising hopes that TRAIL would prove useful as an anti-tumor agent. The production of reliable monoclonal antibodies against TRAIL and its receptors that can stain fixed specimens will allow a thorough analysis of their expression on normal and malignant tissues. Here we report the generation of monoclonal antibodies against TRAIL and its four membrane-bound receptors(TR1-4), which have been used to stain a range of normal and malignant cells, as routinely fixed specimens. Low levels of TRAIL expression were found to be limited mostly to smooth muscle in lung and spleen as well as glial cells in the cerebellum and follicular cells in the thyroid. Expression of the TRAIL decoy receptors (TR3 and 4) was not as widespread as indicated by Northern blotting, suggesting that they may be less important for the control of TRAIL cytotoxicity than previously thought. TR1 and TR2 expression increases significantly in a number of malignant tissues,but in some common malignancies their expression was low, or patchy, which may limit the therapeutic role of TRAIL.Taken together, we have a panel of monoclonal antibodies that will allow a better assessment of the normal role of TRAIL and allow assessment of biopsy material, possibly allowing the identification of tumors that may be amenable to TRAIL therapy.     

Superoxide dismutase in normal and malignant tissues in different species.

1. Superoxide dismutase (superoxide: superoxide oxido-reductase, E.C. 1.15.1.1) in different species was determined quantitatively and qualitatively. Although quantitative differences were minor, there were significant differences in the isoenzyme patterns among the species. 2. No quantitative differences were found in superoxide dismutase (SOD) activities in the brains of mice between 1 and 23 days of age. The mitochondrial isoenzyme increased with age, attaining maximal levels between 9 and 12 days. In the six, regions of adult rat brain studied, highest values of SOD were found in the hypothalamus and lowest in the cortex. 3. SOD levels generally were lower in several transplantable mouse and rat tumors than in normal tissues of these species. Mn-SOD was not detected in the tumors studied by the methods employed.     

Structural differences in ferritins from normal and malignant rat tissues.

Ferritins purified from several normal and malignant rat tissues were examined for amino acid composition, content of tryptic peptides, available sulfhydryl groups and subunit sizes and proportion. Ferritin extracted from adult kidney, neonatal liver and hepatic and renal tumors differed from the ferritin of adult rat liver in migration on electrophoretic gels and in antibody affinity, but did not differ among themselves. Nevertheless, they showed distinctive differences in amino acid composition and tryptic peptide content. All of them and also adult liver ferritin contained two major species of subunits differing in molecular weight. The proportions of subunits, and the available sulfhydryl groups of the intact ferritin molecules, differed among these tissue ferritins. On the basis of amino acid and peptide content, the ferritins of hepatomas and the renal tumor analyzed showec the greatest similarity but not identity. The ferritin of neonatal liver was next most similar. Kidney ferritin differed considerably in composition from tumor and neonatal ferritins, while adult liver ferritin was the most extremely divergent of the series examined. A similar progressive difference was found on examining the proportions of subunits and sulfhydryl groups in these ferritins. However, changes in subunit proportion cannot explain the amino acid and peptide compositional changes.     

Autolysis of phospholipids in homogenates of various plant tissues

The rates of autolysis of endogenous phospholipids were measured in homogenates of 17 cultivated plant tissues. For seven out of the 17 samples, the rates of autolysis of phosphatidylcholine (PC) were greater than 10% hydrolysis per hr (at pH 7.5 and 4°). The addition of dibucaine (2 mM) inhibited the autolytic rates in homogenates of 12 out of 17 samples. The only plant homogenates where dibucaine stimulated the autolytic rates were those of potato tubers. Although dibucaine inhibited the rate of autolysis of PC in potato leaf homogenates there was no advantage to adding it during homogenization and preparation of differential centrifugation fractions from potato leaves. Homogenization with different types and concentrations of osmotica affected the rates of autolysis of PC. Buffering the homogenates at pH 8 drastically inhibited the rates of autolysis in potato leaf homogenates but had little effect on bean leaf homogenates. Various strategies for controlling the rate of membrane lipid breakdown in different plant extracts are discussed.     

Expression of PDE11A in normal and malignant human tissues.

Cyclic nucleotide phosphodiesterase 11A (PDE11A) is the newest member in the PDE family. Although the tissue distribution of PDE11A mRNA has been shown, its protein expression pattern has not been well studied. The goal of this report is to investigate the distribution of PDE11A proteins in a wide range of normal and malignant human tissues. We utilized a polyclonal antibody that recognized all four PDE11A isoforms. Its specificity was demonstrated by Western blot analysis on a recombinant human PDE11A protein and native PDE11A proteins in various human tissues. Immunohistochemistry showed that PDE11A is widely expressed. Various degrees of immunoreactivity were observed in the epithelial cells, endothelial cells, and smooth muscle cells of all tissues examined. The highest expression was in the epithelial, endothelial, and smooth muscle cells of the prostate, Leydig, and spermatogenic cells of the testis, the tubule epithelial cells in the kidney, the epithelial and endothelial cells in the adrenal, the epithelial cells and macrophages in the colon, and the epidermis in the skin. Furthermore, PDE11A expression was also detected in several human carcinomas. Our results suggest that PDE11A might be involved in multiple physiological processes in various organs via its ability to modulate intracellular cAMP and cGMP levels.     

Desmosomal plakophilin 2 as a differentiation marker in normal and malignant tissues

Plakophilin 2 (PKP2) is a widespread protein which shows a remarkable dual location: On the one hand, it appears as a constitutive karyoplasmic protein and on the other it is a desmosomal plaque component of most, probably all, desmosome-possessing tissues and cell culture lines. Here we report on its desmosomal occurrence as revealed by immunocytochemical results obtained with three PKP2-specific murine monoclonal antibodies (mAbs) PP2-62, PP2-86 and PP2-150. These mAbs detect PKP2 in characteristic desmosomes of most normal cells, including simple and stratified epithelia as well as non-epithelial tissues such as myocardium and lymph node follicles. In addition, however, several normal tissues consistently display a differentiation-related PKP2 distribution, for example an absence of immunostaining in the "keratinizing" local specializations of the thymic epithelial reticulum, i.e. Hassall's corpuscles, and the restriction of PKP2 to the stratum basale of most stratified squamous epithelia, in contrast to its absence in upper strata, which contain PKP1- or PKP3-rich desmosomes instead. Taking advantage of the reactivity of mAb PP2-150 with formalin-fixed, paraffin-embedded material, a series of human carcinomas (n = 37) has also been analyzed. The results suggest that mAbs to PKP2 may serve as markers for the identification and characterization of carcinomas derived from--or corresponding to--simple or complex epithelia. Thus consistent PKP2 immunostaining has been observed in all 18 cases of adenocarcinomas tested, but more variable and heterogeneous staining has been noted in squamous cell carcinomas, depending on the specific tumor type. The potential value of such mAbs for cell typing in normal and embryonic tissues and for detecting cell subpopulations with different degrees of differentiation is discussed with respect to their possible application in tumor diagnosis.     

Local uptake and synthesis of oestrone in normal and malignant postmenopausal breast tissues

Uptake and local formation of oestrone (E1) were studied in vivo by a double isotopic technique in normal and malignant breast tissues from 24 postmenopausal women with breast cancer. Active uptake of radio-labelled E1 beyond plasma was found both in normal and malignant tissue, the effect being significantly greater in non-malignant compared with cancer tissue. The presence of local E1 formation was also demonstrated in most samples. Both uptake and synthesis positively correlated with total amount of radioactive E1 found in the tissues. Uptake appeared to make a greater contribution to E1 levels within the breast than in situ synthesis, although there were marked variations between specimens from different patients and the relative proportion of synthesis to uptake was higher in tumour compared with non-malignant tissue. These results demonstrate quantitative differences in the different compartments by which postmenopausal breasts obtain oestrogen and highlight variations between individual breasts. This may be important in optimising oestrogen deprivation therapy for postmenopausal patients with hormone-dependent cancers.     

Distinguishing malignant from normal oral tissues using FTIR fiber-optic techniques

Oral tissue samples were studied using mid-IR fiber-optic attenuated total reflectance spectroscopy and other spectral techniques. The 1745 cm(-1) band, which is assigned to the ester group (C==O) vibration of triglycerides, is a reliable marker that is present in normal tissues but absent or a weak band in malignant oral tissues. Other bands such as C--H stretching bands and the amide bands are also helpful in distinguishing malignant tissues from normal tissues. Subtraction spectra confirmed the above conclusion. In addition, Raman spectroscopic measurements were in agreement with the results observed from FTIR spectra.     

A novel leupeptin-sensitive serine endopeptidase present in normal and malignant rat mammary tissues

Summary N-Methyl-N-nitrosourea (MNU)-induced rat mammary adenocarcinomas contain high levels of a novel leupeptin-sensitive serine endopeptidase. Its properties apparently differ from those of other similar endopeptidases reported to be present in various normal and malignant mammalian tissues. The same leupeptinsensitive serine endopeptidase was also detected in normal rat mammary tissues, but at levels approximately 20 times lower than those in MNU-induced mammary tumors.This enzyme, which is a trypsin-like serine endopeptidase, preferentially hydrolyzes various synthetic endopeptidase substrates at the carboxyl side of an arginyl residue. It has an apparent Mr of approximately 160,000 and a Stokes radius of 49\rA, as determined by gel filtration. Its isoelectric points range from 4.5 to 4.8, and it has a pH optimum of approximately 7.0. The enzyme is stable from pH 4.0 to 7.0, but is extremely unstable above pH 7.0. Besides leupeptin, its activity is inhibited by antipain, aprotinin, N-p-tosyl-L-lysine chloromethyl ketone and phenylmethylsulfonyl fluoride, but is not inhibited by soybean trypsin inhibitor. Many other potential inhibitors or activators such as 2-mercaptoethanol, p-hydroxymercuribenzoic acid and EDTA have no effect on its activity. The enzyme is adsorbed to p-aminobenzamidine agarose affinity beads at pH 6.5 and elutes at pH 4.0.Abbreviations BAPA -N-benzoyl-DL-arginine-4-nitroanilide (all other optically active amino acids in the substrates or inhibitors are of the L-configuration except for the valyl residue in DL-Val-Leu-Arg-4NA) - 4NA 4-nitroanilide - Bz -N-benzoyl - Cbz -N-benzyloxycarbonyl - Suc -N-succinyl - Tos p-toluenesulfonyl - CH₂Cl chloromethyl ketone - DMBA 7,12-dimethylbenz(a)anthracene - MNU N-methyl-Nnitrosourea - Mr apparent relative molecular mass