3H-estradiol and 3H-R5020 binding in cytosols of normal and neoplastic human ovarian tissue

High-affinity cytoplasmic estrogen and progesterone receptors in normal and abnormal ovarian tissues were studied. Estradiol receptor was detectable in 65% and progesterone receptor in 36% of the malignant tumors; 39% of all malignant ovarian tissues were estradiol- as well as progesterone-receptor-positive. Tumors were said to be receptor-positive when the receptors bound greater than 5 fM steroid/mg cytosol protein. No correlations were found between receptor status and histopathological diagnosis. In normal ovarian tissues collected at various phases of the menstrual cycle no changes in [3H]-estradiol and [3H]-R5020 binding could be detected. Analysis of the receptor concentration for both steroid hormones with regard to the menopausal status demonstrated highest levels in postmenopausal women. No significant difference could be found when two groups of patients with advanced ovarian carcinoma associated with the cytosol estrogen receptor status were compared in terms of two different therapeutic schemes (cytosolic chemotherapy with and without tamoxifen).     

The localization of prolactin binding sites in human breast tissue

An immunohistochemical method involving the application of purified human prolactin and a specific antiserum to human prolactin, followed by the peroxidase-anti-peroxidase immunoperoxidase technique, has been used to detect prolactin binding in benign and malignant human breast tissue. The use of fresh, frozen material has been found to be essential. Prolactin binding has been shown to be a consistent feature of benign breast tissue but a variation within samples has been noted which is irrespective of hyperplastic changes. Fifty six percent of breast carcinomas gave a positive reaction but heterogeneity of binding has been shown to be a significant feature. A relationship between the presence and extent of prolactin binding and good histological differentiation of the tumours has been noted. It is concluded that immunohistochemistry is a suitable method for the demonstration of prolactin binding sites in human breast tissue and can provide useful information with regard to tumour heterogeneity.     

Urinary gonadotrophin peptide--isolation and purification, and its immunohistochemical distribution in normal and neoplastic tissues

A urinary gonadotrophin peptide (UGP) was isolated and purified from semi-purified human chorionic gonadotrophin (hCG), prepared from pregnancy urine. The peptide showed hCG-B subunit activity and no hCG-alpha subunit activity as demonstrated by binding studies with the relevant antibodies. It had a molecular weight significantly less than hCG-B subunit. The peptide was linked to thyroglobulin and this conjugate used to immunise rabbits and mice. A radioimmunoassay (RIA) using 125I-UGP and the rabbit antiserum (AK12) was used to monitor chromatographed urine fractions from patients with ovarian carcinoma, seminoma and hydatidiform mole. UGP was also found in the urine extract of a healthy male, but at a much lower level. In each case the UGP detected had the same molecular weight as the pregnancy preparation and appeared to be the main gonadotrophin constituent in those urine samples. Initial immunohistochemical screening of normal and neoplastic tissues with the rabbit antibody (AK12) showed reactivity with some tumours including carcinomas of the lung, ovary, cervix and breast as well as trophoblastic and germ cell tumours. Reactions with non-neoplastic tissues were confined to some specialised epithelia and macrophage populations. A more comprehensive immunohistochemical study was made using a monoclonal antibody to UGP (2C2), with a monoclonal antibody to conformational hCG (INN 13) and another monoclonal antibody to free B subunit (1E5) as controls. Similar patterns of reactivity were produced by the AK12 and 2C2 antibodies in both neoplastic and non-neoplastic tissues. Additional tissues were investigated with the three monoclonal antibodies. The 2C2 antibody reacted with 93% (77/83) of tumours examined; the INN 13 antibody reacted with only the syncytiotrophoblast cells of choriocarcinoma, hydatidiform mole, placental site trophoblastic tumour, and in one case of seminoma; the 1E5 reactivity was confined to only choriocarcinoma syncytiotrophoblast cells.     

Insulin binding by normal and neoplastic colon tissue

Insulin receptors in human colon tumours, normal colon tissue and mesenteric fat removed at surgery have been identified by measuring the binding of labelled insulin to cell membrane preparations. Insulin binding sites were readily detected in all tissues, with mean +/- SD binding site concentrations of 43 +/- 41, 44 +/- 39, and 44 +/- 35fmol/mg membrane protein, and dissociation constants of 0.73 +/- 0.61, 0.66 +/- 0.41, and 0.78 +/- 0.58nM for microsomal plasma membrane preparations of tumour (n = 23) respectively. The specificity of binding of labelled insulin was similar in tumour and normal colon samples. Binding in normal colon preparations was highest in the epithelium (84fmol/mg membrane protein) and lower in lamina propria (19fmol/mg), submucosa (25fmol/mg) and muscle wall (13fmol/mg). Degradation of labelled insulin was similar in tumour and normal colon preparations. Mean receptor levels were not significantly different between microsomal membrane preparations and plasma membranes partially purified on discontinuous sucrose gradients, quantitated against either unit membrane protein or unit 5'-nucleotidase specific activity. There was a significant negative correlation between insulin levels, but no significant relationship was seen between serum insulin and receptor levels in either colon tumour or tissue preparations from full-thickness normal colon wall. An inverse correlation between serum insulin and receptor levels was, however, apparent in preparations of colonic musosa. These data indicate that although insulin receptors in colon tumours share the same biochemical characteristics as those present in the normal colon, receptors in tumour tissue are less sensitive to down-regulation by ambient insulin than receptors in mesenteric fat cells and normal colonic mucosa.     

Distribution of antiestrogen-specific binding sites in normal and neoplastic mammary gland

Recently, several investigators have demonstrated the presence of triphenylethylene antiestrogen binding sites in the cytoplasm of many tissues, which specifically bind to radioactive tamoxifen with high affinity. Although mammary gland is one of the principal target organs for antiestrogen action, the characterization of antiestrogen binding in mammary tissues has not been reported. We have studied the antiestrogen binding properties of [3H]tamoxifen in the mammary glands of virgin, pregnant and lactating rats as well as in the N-methyl-N-nitrosourea-induced mammary tumors. Tritium labeled tamoxifen bound specifically and with high affinity (Kd = 10(-9) M) to components present in 25,000 g supernatant. Unlabeled estradiol or DES did not compete for these sites, whereas unlabeled tamoxifen showed competitive inhibition. The mammary glands contained threefold higher levels of cytoplasmic binding sites as compared to the mammary tumors. Mammary glands from the pregnant rats bound tamoxifen to a greater extent than that of either virgin or lactating rats. The functional relevance of these binding sites is still unknown.     

Total prolactin binding sites in human breast cancer biopsies

Summary Total and free prolactin receptors were assayed in 72 human breast tumor biopsies. Forty-nine percent of the tumors are positive (specific binding greater than 0.8% of total radioactivity) when assaying free receptors, while 71% are positive when total receptors levels are determined. No clear relationship exists between the prolactin receptor positivity and the presence of either progesterone or estradiol receptors. Measurement of total prolactin receptors could be an important and independent criterion of the hormonal sensitivity of the tumors. Address for reprints: Dr. J.P. Peyrat, Laboratoire d'Endocrinologie Expérimentale, Centre de Anticancéreux de Lille (Centre Oscar Lambret), BP 307, 59020 Lille Cédex, France.     

Differential immunohistochemical localization of xanthine oxidase in normal and neoplastic human breast epithelium

Xanthine oxidase (XO) and xanthine dehydrogenase (XDH) are alternate enzymatic forms of the XO/XDH protein that catalyzes the oxidation of hypoxanthine to xanthine, and xanthine to uric acid, and in the process XO/XDH generates reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and hydroxyl radicals. We hypothesize that XO/XDH, which is expressed in mammary epithelium, contributes to the development of breast cancers by virtue of its ability to generate genotoxic ROS. In this study, we produced human XO/XDH protein at high levels in Spodoptera frugiperda (Sf9) insect cells using the baculovirus vector to confirm the specificity of antibodies used for immunostaining of human breast tissues. Immunoblot analysis demonstrated that the full length 143 kDa polypeptide was partially processed into a 87 kDa and 59 kDa fragments. The overexpressed XO/XDH protein was identified in the cytoplasm of insect cells by immunofluorescence staining. Using these antibodies we analyzed normal and neoplastic breast epithelium for the presence of XO/XDH. Immunohistochemical analysis of normal human breast revealed the presence of XO/XDH in the cytoplasm of epithelium lining terminal ducts. The intensity of XO/XDH staining was markedly enhanced in alveolar epithelium of lactating mammary lobules. In contrast, no immunohistochemically detectable XO/XDH was observed in intraductal in situ carcinomas and in invasive carcinomas of the breast. Further studies are necessary to confirm the utility of the loss of XO/XDH expression as a marker for neoplastic change in the breast and investigate the functional role of this enzyme in the pathogenesis of breast cancer.     

Glutathione transferase isoenzymes in normal and neoplastic human kidney tissue.

Glutathione transferase (GST) activity in the cytosolic fractions of renal cortex tumour was found to be significantly lower (215 +/- 156 mU/mg) than that present in the corresponding non-tumour (466 +/- 278 mU/mg) tissues. Using the immunoblotting technique, glutathione transferase isoenzymes expression in both tumour and non-tumour kidney was investigated. Alpha and pi class glutathione transferases were the most abundant enzymes in non-tumour kidney and were expressed by all samples investigated. Immunofluorescence analysis indicated that the pi class enzymes are localized mainly in the distal convoluted tubules, whereas alpha class enzymes are localized in the proximal tubules. In the tumour moiety the alpha class GST appears to be absent or expressed at low level as compared with non-tumour samples. On the contrary, no significant differences in the expression of pi class GST were found in tumour as compared with non-tumour tissues. Mu class GST protein was detected in 12 of 26 samples tested. When present, mu class GST constitutes a few per cent of total GST protein. Immunofluorescence studies indicate that mu class GSTs are localized within the distal convoluted tubules. According to the electrophoretic mobility at least two different mu GST subunits (26.5 and 27.5 kd) were found. In one sample only the faster mu class GST subunit was present, two samples expressed both types of GST subunits, whereas nine samples expressed only the slower GST subunit. With the exception of one sample, a reduction of mu class GST expression was seen in tumour as compared with non-tumour tissues. The decrease of activity seen in the cytosolic fraction of tumour kidney must be ascribed mainly to a reduction or to a lack of expression of alpha class GST and to a lesser extent of mu class GST.     

Cyclin nucleotide binding sites of the smooth endoplasmic reticulum from normal and neoplastic liver in the rat.

The studies are presented which demonstrate that smooth endoplasmic membrane of normal liver has a single apparent binding site for cAMP with a KD of 0.6 X 10(-8) M. In contrast to this, however, cyclic AMP binding to the intracellular membrane of hepatoma 7800 exhibit two binding sites; the binding constant of one site on the tumor membrane is comparable to that of the normal liver whereas the value of the second intrinsic association constant differ by a factor of 10. It is suggested that there may be an association between abnormal cyclic nucleotide metabolism and the intracellular membrane modulation of the expression of genetic information in normal and neoplastic cells.     

Comparison between concentrations of trace elements in normal and neoplastic human breast tissue

Histologically normal and neoplastic human breast tissues obtained from 25 patients at the time of mastectomy were homogenized (200 mg/ml) in distilled water and 5-microliter aliquots dried on Formvar films for trace element analysis by energy-dispersive X-ray fluorescence. The elements measured were calcium, vanadium, copper, zinc, iron, chromium, manganese, nickel, selenium, molybdenum, bromine, rubidium, strontium, mercury, arsenic, and lead. In general, significantly large increases (p less than 0.001) in calcium, vanadium, copper, zinc, selenium, and rubidium were found in breast tumors, with a less significant increase (p less than 0.05) for nickel. When a comparison was made between histologically normal and neoplastic tissues from the same individual, zinc and rubidium were found to be consistently higher in the tumor, whereas calcium, copper, and vanadium levels varied from normal to high. In no instance were the tissue changes in calcium, copper, zinc, or rubidium reflected in the blood levels, which were within normal limits. The distribution of calcium, copper, and zinc in urine varied among individuals with primary tumors; however, rubidium levels tended to be consistently elevated. An attempt is being made to correlate these various differences with the extent of the primary disease at the time of surgery, the postoperative tumor-free interval, and subsequent therapy.     

Hyperthermia-induced vascular injury in normal and neoplastic tissue

The sequential morphologic alterations in normal skeletal muscle in rats, Walker 256 tumors in rats, and transmissible venereal tumors (TVT) in dogs following microwave-induced hyperthermia (43 degrees C and 45 degrees C for 20 minutes), were studied by histologic and ultrastructural examination. Normal muscle and Walker 256 tumors showed edema, congestion, and hemorrhage at 5 minutes post-heating (PH), followed by suppuration, macrophage infiltration, and thrombosis at 6 and 48 hours PH, and finally by regeneration and repair by 7 days PH. Vascular endothelial damage and parenchymal degeneration were present 5 minutes PH. Progressive injury occurred for at least 48 hours PH. Two hyperthermia treatments separated by a 30- or 60-min cooling interval, were applied to Walker 256 tumors in a subsequent study. Increased selective heating of tumor tissue versus surrounding normal tissue, and increased intratumoral steady state temperatures were found during the second hyperthermia treatment. Canine TVTs were resistant to hyperthermia damage. These results suggest that vascular damage contributes to the immediate and latent cytotoxic effects of hyperthermia in normal tissue and some types of neoplastic tissue, and that selective heating of neoplastic tissue occurs in tumor tissue with disrupted microvasculature.     

Confocal endomicroscopic imaging of normal and neoplastic human tongue tissue using ALA-induced-PPIX fluorescence: a preliminary study

Confocal endomicroscopy is a novel and non-invasive microscopic technique that enables surface and subsurface imaging of living tissues or cells in vivo. The purpose of this study was to assess the possibility of utilizing a rigid confocal endomicroscope (RCE) system developed for detecting morphological changes in living normal and neoplastic human tongue tissue in combination with 5-aminolevulinic acid (ALA)-induced endogenous protoporphyrin IX (PPIX) fluorescence. Three patients with squamous cell carcinoma (SCC) of the tongue were examined using the novel RCE system with the excitation wavelength at 488 nm from an argon-ion laser and the detection wavelengths of the tissue fluorescence above 515 nm. Patients were topically applied with 0.4% of 5-ALA rinsing solution to the oral mucosa for approximately 15 min, and then the confocal endomicroscopic imaging of tissue PPIX fluorescence was performed on the lesion sites of the tongue after an optimal incubation period of 90-120 min. For comparison purposes, ALA-PPIX fluorescence confocal endomicroscopic imaging was also carried out on the normal sites of the tongue in vivo from two healthy volunteers. Image distortions due to tissue motion can be minimized using a specially designed tissue stabilizer attached to the RCE probe. Good quality ALA-mediated confocal fluorescence images of the tongue can be acquired in real-time, providing well-defined micro-morphological structures (e.g., filiform papillae, keratinized epithelium and fungiform papillae) of the tongue in vivo. Changes of tissue structures in oral tissue associated with cancer transformation can also be clearly identified using the RCE imaging. Preliminary results obtained in this study suggest that ALA-mediated rigid confocal endomicroscopy may have a significant potential for the rapid, non-invasive diagnosis and evaluation of early oral cancers in vivo.