In vitro growth promotion of rat leukemia L5222 cells in the presence of 2-mercaptoethanol.

Propagation of the transplantable acute rat leukemia L5222 in vitro was highly dependent on the reducing agent 2-mercaptoethanol (2-Me). Maximum growth promotion was obtained in the simultaneous presence of 1.25-2.5 x 10-4m 2-Me and 30% fetal bovine serum in enriched tissue culture medium NCTC 135. A short exposure to the reducing agent insured growth of the leukemia cells for 5 days, but extended culture necessitated constant presence of the thiol. After 2-8 days of culture, inoculation of the cell suspension produced widespread leukemia in inbred BD IX rats.

Modulation of plasminogen activator systems by matrix components in two breast cancer cell lines: MCF-7 and MDA-MB-231.

We have analyzed the plasminogen activator (PA) systems of two metastatic breast adenocarcinoma cell lines, MCF-7 and MDA-MB-231, as a function of 17 beta-estradiol stimulation when the cells were cultured on purified components of extracellular matrix. Laminin enhanced PA levels in both cell lines, but this enhancement seemed to occur via different mechanisms, including dissociation of inhibitor complexes. The major effect was the marked increase in cell-associated urokinase-type PA (u-PA); the increase was independent of estrogen in hormone-insensitive MDA-MB-231 cells grown on laminin-coated surfaces. In estrogen-sensitive MCF-7 cells, 17 beta-estradiol stimulated u-PA secretion in a similar fashion on plastic, laminin, fibronectin, or collagen but acted in synergy with laminin in the production and release of tissue-type PA.

Estrogen receptor immunocytochemical assay (ER-ICA): computerized image analysis system, immunoelectron microscopy, and comparisons with estradiol binding assays in 115 breast carcinomas.

An estrogen receptor (ER) immunocytochemical assay (ER-ICA) was applied to 115 malignant breast carcinomas and the results were compared to those of steroid binding assays performed on cytosol extracts of the same tumors. Immunoperoxidase (peroxidase-antiperoxidase) staining was performed on frozen sections using rat monoclonal antibody to estrogen receptor H222SP gamma. A preembedding method was used for the immunoelectron microscopy study. A semiquantitative analysis and a computerized image analysis system (SAMBA 200 TITN) were used to evaluate the positive ER immunostaining. Positive immunostaining (81 of 115) was always located in the nucleus of tumor cells and of normal cells in adjacent breast tissue. The immunostaining pattern differed from one tumor to another, due to variations in either the intensity or the percentage of positive cells. When immunohistochemical staining was correlated to biochemical assay, there was an 88% correlation, and staining intensity and percentage of positive cells significantly increased (P less than 0.01) with cytosolic ER levels and were independent of cellularity. These results indicated that ER-ICA is to date the most reliable histochemical method for ER detection and correlated in 88% of the cases with ER biochemical assay; ER-ICA constitutes a method particularly valuable to screen ER negative tumors on condition that tumor fragment quality (sampling and storage) is perfectly controlled; ER-ICA provides additional information for heterogeneous ER distribution within tumors; ER-ICA as a qualitative method is unable to replace the quantitative ER determination obtained with biochemical assay although the computerized system (SAMBA 200) for image analysis of microscopic preparations constitutes a valuable improvement of immunostaining analysis; and ER-ICA based on ER antigenic site detection is complementary to biochemical assay based on ER functional site determination.

Concomitant secretion by A431 cells of tissue plasminogen activator and a specific inhibitor masks EGF modulation of tPA activity.

It has previously been reported that EGF enhances uPA but not tPA in the A431 squamous carcinoma cell line. To determine whether the absence of tPA modulation by EGF reflected steady levels or the action of an anti-activator, we assayed tPA, PAI-1 and tPA/PAI-1 complexes by zymography and immunological assays. Under conditions in which EGF had no effect on tPA activity, tPA antigen paradoxically increased with a concomitant rise of tPA/PAI-1 complexes. This indicated that tPA was rapidly inactivated through the formation of a complex, immunologically and electrophoretically related to tPA/PAI-1. tPA antigen and tPA/PAI-1 complexes were modulated by EGF in a time and concentration dependent manner. PAI-1 antigen was secreted into A431 medium (CM) after a lag phase of 16 h in both control and EGF-treated cultures. Evidence is presented here that two forms of PAI-1 are present in A431 CM: an inactive form and an active form which neutralizes the tPA secreted, masking its enhancement by EGF in functional assays.

Early alterations at the plasma membrane of breast cancer cell lines in response to estradiol and hydroxytamoxifen.

The time course of the early stage of estradiol-17 beta (E2) and hydroxytamoxifen (OHTAM) action at the plasma membrane of hormone-responsive MCF-7 and non-responsive MDA-MB-231 (MDA) breast cancer cell lines was investigated using scanning electron microscopy (SEM), electron probe X-ray microanalysis and microelectrophysiology analysis. SEM showed a marked increase in the density and the length of microvilli (MV) on MCF-7 cells treated with 1 nM estradiol for 1 min. This membrane response disappeared at 5 min. No early effect was obtained with OHTAM, but both compounds produced a similar surge of heterogeneous MV at 15 min of treatment. The morphological change induced by E2 subsided at 60 min, whereas that of OHTAM persisted. X-ray microanalysis and computer determination of peak/background ratios permitted the demonstration that these morphological alterations were concomitant with a rise in the intracellular level of potassium. Microelectrophysiology analysis showed a sharp transitory decrease in the membrane potential of MCF-7 cells in response to estradiol. In the estrogen-insensitive MDA cells, the hormone did not modify the membrane potential and K levels decreased at 1 and 5 min before rising again to control levels at minute 15 when MV appeared. With OHTAM, potassium decreased significantly at 60 min of treatment. These initial and transitory changes in surface morphology paralleled by alterations in potassium level may be consistent with the occurrence of estrogen membrane receptors on target cells, a new aspect of steroid hormone action.

Immunohistochemical detection of laminin in 98 human breast carcinomas: a light and electron microscopic study.

The distribution of laminin was studied in 98 breast carcinomas with antilaminin and the avidin-biotin-peroxidase complex method. Laminin was observed within vascular and epithelial basement membranes. Laminin displayed a continuous linear pattern in intraductal carcinomas, and it was heterogeneously distributed, with a discontinuous linear pattern, in invasive carcinomas. No intracellular laminin staining was detected. Electron microscopic study showed laminin immunostaining in the lamina densa of basement membranes in nonneoplastic breast tissue. In tumors, laminin immunostaining frequently revealed multilayered basement membranes and abnormal multilayered basement membranes in blood vessels in the tumor stroma. These data suggest that laminin immunostaining, as a new approach to the heterogeneous basement membrane changes occurring in carcinomas, should permit better understanding of cell diffusion processes and of stroma-tumor cell interactions. The consistent extracellular distribution of laminin in contact with the stroma indicates that the latter plays an important role in the assembly of basement membrane components.

Modulation of tPA, PAI-1 and PAI-2 antigen and mRNA levels by EGF in the A431 cell line.

It has been reported that EGF treatment enhances uPA but not tPA in the A431 epidermoid carcinoma cell line. To determine whether the absence of tPA modulation by EGF could be due to the action of inhibitors, we assayed tPA, PAI-1, PAI-2 and tPA/PAI-1 complexes by immunological assays and zymography in A431 serum-free medium. We found that, under conditions in which EGF had no effect on tPA activity, tPA antigen increased with a concomitant rise of tPA/PAI-1 complexes, indicating the action of an inhibitor. Both tPA antigen and tPA/PAI-1 complexes were modulated by EGF in a time and concentration dependent manner. tPA/PAI-1 complex levels were lower than tPA levels, suggesting the presence of other inhibitors. Immunological assays detected PAI-2 in addition to PAI-1 and showed a time and dose response to EGF. Modulation of tPA and the anti-activators by the growth factor was confirmed by identification of the corresponding transcripts with cDNA probes. We conclude that the net plasminogen activator activity in A431 cells is the result of a balance between activators and inhibitors.

[Breast tissue: estrogen receptors. Immunohistochemical and biochemical analysis of normal and hyperplastic tissue and carcinoma in situ. Apropos of 86 cases]. / Tissu mammaire: récepteurs estrogéniques. Analyse immunohistochimique et biochimique du tissu normal, hyperplasique et du carcinome in situ. A propos de 86 cas.

Estrogen receptors (ER) were investigated using immunohistochemical techniques in 54 cases and biochemical techniques in 38 cases on a series of biopsy specimens of normal, hyperplasic and malignant mammary tissue (intraductal carcinoma). Immunohistochemical data were submitted to quantitative and semi-quantitative analysis (SAMBA 200, TITN). On normal tissue, immunostaining is bright and evenly distributed in galactophores and ductulo-lobular junctions; it is unevenly distributed but consistently present in lobules and increasing after menopause. On hyperplasic tissue, immunostaining of papillary cystadenomas and epitheliosis and fibroadenosis zones was similar to normal tissue with respect to the intensity and heterogeneity. Conversely, their immunohistochemical features are close to blunt duct adenosis and atypical lobular hyperplasia of ductulo-lobular junctions. On malignant tissue, immunostaining is unpredictable, it can be evenly distributed, unevenly distributed in patches or gradients, or totally absent. Immunohistochemical techniques can only be used to assess the intensity and distribution of ER in function of cell type in normal tissue and during a cancerous process. Thus strong receptivity of ductulo-lobular junctions may reinforce the claim that these structures are the targets for estrogen co-carcinogenic substances. The heterogeneity of ER at the onset of carcinoma suggests the possibility of several pathway of development. The fact that myoepithelial cell never seem to display estrogen type receptivity makes it logical to seek a special sensitivity for them.

Laminin immunodetection in tumorous and nontumorous disorders of human thyroid.

Laminin distribution in tissues from surgically removed thyroids consisting in normal gland (5), Basedow's disease (5), thyroiditis (5), follicular adenomas (8), papillary carcinomas (8), follicular carcinomas (6) was studied using rabbit laminin antibody raised against murine laminin. Immunoperoxidase technique (Avidin-Biotin-Peroxidase Complex) was performed on (1) paraffin sections of fixed tissue (2) frozen sections for light microscopy examination. Vibratome thick sections (100 micron) and pre-embedding technique were used for electron microscopy study. Positive staining, was obtained only on frozen and vibratome sections and was found within basement membranes but never in epithelial cell cytoplasm. Laminin had a similar distribution in follicular adenomas, Basedow's disease and normal tissue. Nests of damaged cells in Hashimoto's thyroiditis lacked positive laminin immunostaining. In papillary carcinomas positive staining was found beneath the epithelial cells along the cores. In well differentiated follicular carcinomas the perifollicular laminin staining was preserved, whereas in poorly differentiated follicular carcinomas laminin staining was barely visible or absent.

[Invasion, metastases of solid tumors. Interaction of tumor cells with tissue and vascular basement membranes]. / Invasion, métastases des tumeurs solides. Interactions cellules tumorales/membranes basales tissulaires et vasculaires.

Since early antiquity malignant tumors have been recognized and characterized by: 1) their particular local-regional invasive properties. Developing irregularly from the primary tumor mass, the invasion of adjacent tissues often displays classical crab-like images which distinguish, macroscopically and microscopically, malignant tumors from benign tumors. 2) their metastatic capacities. Even though they have been taught since the time of Hippocrates, the specific clinical characteristics of malignant tumors have only recently been apprehended and studied on cellular and biochemical levels. The development of the metastatic phenomenon is a complex chain of events limited to only a few tumor subpopulations. Like every biological study knowledge has progressed over the past years only by concentration of efforts on some of these stages: Invasion associated with disorganization and destruction of basement membranes; Migration of metastatic tumor cells in the stroma and bridging of vascular basal membranes; Colonization of target tissues by recognition and penetration of specific vascular membranes, establishment and concentric development within these tissues. These three stages result from successive interactions between multiple cellular and biochemical phenomena. This review is a schema of our present knowledge of tumor invasion, and more precisely of that concerning the relations between tumor cells and extracellular matrices, particularly basal membranes, leading to a definition of the cellular phenotype with a high metastatic potential.

Estrogen receptor immunocytochemical assay (ERICA) and laminin detection in 130 breast carcinomas and computerized (Samba 200) multiparametric quantitative analysis on tissue sections.

An estrogen receptor immunocytochemical assay (ER-ICA) was applied to 130 malignant breast carcinomas and the results were compared to those of steroid binding assays performed on cytosol extracts of the same tumors. Also laminin (lam) distribution was studied in the same tumors. A semi quantitative analysis and a computerized image analysis system (SAMBA 200 TITN) were used to evaluate the positive ER and lam immunostaining. Positive ER immunostaining was always located in the nucleus of tumor cells and of normal cells in adjacent breast tissue. When immunohistochemical staining was correlated to biochemical assay there was a 88% correlation staining intensity and percentage of positive cells significantly increased (p less than 0.01) with cytosolic ER levels. Lam was observed within vascular and epithelial basement membranes (BMs). Lam staining displayed a continuous linear pattern in intraductal carcinomas or was heterogeneously distributed with a discontinuous linear pattern in invasive carcinomas. No intracellular lam staining was detected. In tumors, laminin immunostaining revealed often multilayered BMs and abnormal multilayered BMs in blood vessels in the tumor stroma. These results indicated that (ER-ICA) is to date the most reliable histochemical method for ER detection and correlated in 88% of the cases with ER biochemical assay ER-ICA provides additional information for heterogeneous ER distribution within tumors ER-ICA as a qualitative method is unable to replace the quantitative ER determination obtained with biochemical assay ER-ICA based on ER antigenic site detection is complementary to biochemical assay based on ER functional site determination laminin immunostaing constitutes a new approach to the heterogeneous BM changes occurring in carcinomas, and permits a better understanding of cell diffusion processes and of stroma-tumor cells interactions: the consistent extracellular lam distribution in contact with the stroma, indicates that the latter plays an important role in the assembly of BM components the SAMBA 200 permits a reliable accurate evaluation of the percentage of the immunostained cells and surfaces.

[Laser therapy in the prevention and treatment of mucositis caused by anticancer chemotherapy]. / La laserthérapie dans la prévention et le traitement des mucites liées à la chimiothérapie anticancéreuse.

The appearance of mucositis is a frequent and painful secondary effect of anticancer chemotherapy. Patients who develop oral toxicity during the first course of treatment will almost assuredly show identical side effects during each subsequent course unless the drugs are changed or the doses are lowered. In the absence of an efficacious antidote or preventive prophylaxis for such lesions to date, this report presents the results of a preliminary retrospective non-randomized study of the effect of soft-laser treatments on mucositis in cancer patients receiving combination chemotherapy, including 5-fluorouracil. Iatrogenic mucositis was observed during 43% of 53 chemotherapy cycles in the case control population. Curative laser therapy reduced the time to repair lesions and the rate of therapeutic modifications. For patients who received soft-laser therapy as a preventive measure, the incidence of oral complications was reduced to 6% during 101 cycles of chemotherapy. All of these patients, even those who have encountered mucositis before receiving preventive laser therapy, terminated their cancer therapy as originally scheduled. Well designed and carefully controlled trials will be necessary to define the place of helium-neon laser therapy in the repair and prevention of oral complications due to cancer chemotherapy.

[Immunocytochemical detection of laminin by light and electron microscopy: study of changes in the basement membrane in tumor pathology]. / Détection immunocytochimique de la laminine en microscopie optique et èlectronique: ètude des modifications de la membrane basale en pathologie tumorale.

A wide range of normal human tissue samples including cervix, endometrium, thyroid, pancreas, parotid, breast, placenta, gastric mucosae, striated muscle were compared with tumorous and non tumorous disorders (thyroiditis, Graves disease, follicular adenoma, thyroid carcinomas, breast cystic disease, fibroadenoma, adenosis, breast carcinomas) using anti-laminin and Avidin Biotin Peroxidase complex method on frozen sections (light microscopy study) and vibratome cut 100 micrometer-thick-sections (electron microscopy study). It was shown that laminin was located in the lamina densa of basement membranes (BM) in normal human tissue and visible on BM like structures around decidua cells, BM were abnormally thick and often multilayered but continuous and laminin positive in intraductal breast carcinomas and well differentiated follicular carcinomas of thyroid, in invasive carcinomas laminin immunostaining displayed an heterogeneous pattern with disruptions and even may completely disappeared, in tumor stroma, blood vessels BM had a laminin abnormal staining with a multilayered pattern. Since laminin is involved in cell attachment to basement membrane through specific receptors to laminin and to biochemical components of modified interstitium found in tumorous disorders, laminin immunohistochemical detection constitutes a valuable method for a better understanding of tumor cells diffusion and metastases development.

[Dosage and modulation of cutaneous steroid receptors during antihormonal therapy in a case of Stewart-Treves syndrome]. / Dosage et modulation des récepteurs stéroïdiens cutanés sous thérapeutique anti-hormonale dans le cas d'un syndrome de Stewart-Trèves.

We report a case with clinical symptoms of a Stewart-Treves syndrome 2 years after homolateral mastectomy for carcinoma. The mammary origin of the syndrome was found at histology and confirmed by assays of hormone receptors in skin biopsies before and during anti-hormone therapy. This treatment rapidly attenuated the clinical signs of both the lymphedema and the pseudo-Kaposi cutaneous lesions. Hormone receptor determinations are suggested as useful tools for the diagnosis and for surveillance of therapeutic efficacy. CASE HISTORY. This 68-year old patient whose right breast was removed in April, 1985 for scirrhous carcinoma was hospitalized in February, 1987 for lymphedema, pain and functional impotence of the right arm accompanied by Kaposi-like skin lesions in the homolateral mammary region, the right shoulder and the upper third of the right arm. Tamoxifen administration in doses of 30 mg/day was started on the day of the first biopsies at sites of the cutaneous lesions and continued for 60 days. Additional biopsies were taken on days 30 and 60 of therapy. Under therapy clinical improvement was noted with decrease in the lymphadenopathy and in the size of the nodular skin lesions. PATHOLOGY AND STEROID HORMONE ASSAYS. Standard histological studies were performed on one of the biopsies. The remaining tissues were flash frozen and assayed for estrogen and progesterone receptors. ER was determined by radio-ligand assay (RLA) to measure the free binding sites of the hormone or anti-hormone and by enzyme immunoassay (EIA) to detect both free and occupied antigenic sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose-related enhancement of erythropoiesis by sulfhydryl compounds and its reversal with a thiol inhibitor.

Erythropoietin-mediated erythrocyte development from bone marrow of hypertransfused rats was significantly greater when the culture medium contained an optimal dose of certain sulfhydryls. This stimulatory action was attributed to the presence of SH groups because erythroblast numbers fell to control levels when the culture contained the thiol inhibitor, p-hydroxymercuribenzoate.