Differential glucocorticoid effects on proliferation and invasion of human trophoblast cell lines.

Several clinical situations require continuous glucocorticoid (GC) treatment during pregnancy. A well-known deleterious side effect of such treatment is the higher incidence of growth-restricted fetuses, for which a too shallow trophoblast invasion is presently hypothesised as the underlying cause. This study investigated whether the synthetic GC triamcinolone acetonide (TA) influences proliferation, invasion and endocrine activity of human trophoblast. BeWo and JEG-3 choriocarcinoma cell lines both express GC receptors (western blotting) and were used as models for human trophoblast. JAR devoid cells of GC receptor were used as negative control. The cells were cultured for 48 h without (control) or with 0.5, 5 and 50 microM TA. In the presence and absence of serum, proliferation was determined by cell counting and measuring the cell cycle regulating protein cyclin B1 (Western blotting); invasion was determined by a conventional Matrigel invasion assay and by measuring the secretion (ELISA) of matrix-metalloproteinases (MMP-2, MMP-9) into the culture medium; endocrine activity was assessed by measuring the levels of human chorionic gonadotropin (ELISA) into the culture medium. TA altered the number of viable and dead cells as well as cyclin B1 levels and, to a lesser extent, invasion of BeWo and JEG-3, with a strong influence of serum. BeWo and JEG-3 cells reacted differently and in most instances reverse. In the cell lines used as models of human trophoblast, TA alter some functions relevant to proliferation and invasion, and suggest that caution should be exercised when treating women with GCs during pregnancy.

Serum-dependent effects of IGF-I and insulin on proliferation and invasion of human first trimester trophoblast cell models.

Extravillous cytotrophoblasts are specialised epithelial cells of the placenta that proliferate or invade the maternal decidua. Little is known about the mechanisms that regulate these processes. Here the effects of several insulin and insulin-like growth factor-I (IGF-I) doses, either singly or in synergy with serum, on human chorionic gonadotropin-beta (hCG-beta) secretion (RIA), proliferation (cell counting, cyclin B(1) levels) and invasion [Matrigel invasion assay, secretion of matrix metalloproteinases (MMP) 2 and 9] were investigated. The choriocarcinoma cell lines BeWo, JAR and JEG-3 served as models for first trimester human trophoblasts. Both growth factors altered hCG-beta secretion and proliferation dependent on the cell line. Insulin stimulated proliferation in JAR cells and, to a lesser extent, in JEG-3 cells, and when cultured in serum-free medium, BeWo was not affected. Invasion was not affected although proMMP-2 levels in culture medium were altered under some conditions. A strong synergistic effect with serum was noted. In the presence of serum both growth factors reduced proliferation and invasion in a similar fashion. Since the cell models differ by their degree of differentiation, the data demonstrate that the effects of insulin and IGF-I strongly depend on serum and the degree of differentiation. It can be speculated that IGF-I can take on tasks of insulin in the regulation of trophoblast functions under conditions of insulinopenia.

Mercurochrom can be used for the histochemical demonstration and microphotometric quantitation of both protein thiols and protein (mixed) disulfides.

Mercurochrom [2,7-dibromo-4-(hydroxymercuri)-fluorescein disodium salt] used for staining of protein thiols in addition binds to other groups of proteins. Experimental evidence is provided that mercurochrom bound to non-thiol groups forms a 1:1 adduct with protein (mixed) disulfides. The disulfide contents of three different types of cells determined biochemically correlated with the corresponding mean integrated optical densities determined microphotometrically after mercurochrom staining of groups other than thiols. Intracellular disulfide exchange has been studied, leading to a transformation of protein mixed disulfides to protein disulfides and an equimolar loss of protein thiols. Protein mixed disulfides were generated from protein thiols using both methyl methanethiosulfonate (MMTS) and 2,2'-dihydroxy-6,6'-dinaphthyldisulfide (DDD). Loss of thiols as well as the equimolar increase of protein mixed disulfides were followed using both mercurochrom staining for thiols and for disulfides. Generation of protein mixed disulfides due to the DDD reaction was also followed by azocoupling with Fast blue B. On the basis of the observed stoichiometry between the loss of protein thiols and the quantity, increase or conversion of protein disulfides determined microphotometrically using both mercurochrom staining and DDD Fast blue B staining, we conclude that: (1) 1 mol of mercurochrom is bound per mol of protein (mixed) disulfide; and (2) the molar absorptivity of mercurochrom bound to disulfides is epsilon 520 = 34940. This study demonstrates that mercurochrom can be used for the quantitative determination of the oxidative status of protein thiols in cells.

The quantification of the histochemical protein staining with 2-hydroxy-1-naphthaldehyde (HNA) demonstrating primary amino groups of proteins.

The usefulness of the HNA-pH4-1d staining, which histochemically demonstrates primary protein amino groups under the conditions used, for the microphotometric quantification of proteins was investigated. A correlation (r = 0.986) has been found between the mean protein contents of fresh frozen and fixed sections prepared from different tissues of rats and the corresponding mean integrated extinction values determined histophotometrically after HNA-pH4-1d staining. A histophotometric extinction of E = 0.284 corresponded to 10(-12) g protein. The mean integrated extinction values determined cytophotometrically of different single cells and nuclei stained using the tetrazonium coupling method for proteins correlated (r = 0.989) with corresponding extinction values measured after HNA-pH4-1d staining. A cytophotometric extinction after HNA-pH4-1d staining of E = 0.130 correspond to 10(-12) g protein.

Quantitative measurements of the changes in protein thiols in cervical intraepithelial neoplasia and in carcinoma of the human uterine cervix provide evidence for the existence of a biochemical field effect.

Quantitative micromethods have been used for measuring reactive protein thiols (PSHr), total reactive protein sulfur (TRPS), total protein thiols (PSHt), and protein disulfides (PDS) in fixed frozen sections of human uterine cervix. PSHr and TRPS were stained using 2,2'-dihydroxy-6,6'-dinaphthyl disulfide; PSHt and PDS were stained using mercurochrome methods. Microspectrophotometric measurements were made on the stained sections using a microdensitometer with associated data processing; the results obtained for areas of epithelium and stroma were converted to absorbance values per micron 2. Samples of uterine cervix that were diagnosed as containing cervical intraepithelial neoplasia (CIN) I-III or carcinoma were examined and compared with samples of normal uterine cervix. Measurements were made not only on identified lesions but also on apparently normal tissue obtained from the same cervix. Epithelial/stroma ratios (E/S) were calculated for PSHr, TRPS, PSHt and PSHt + PDS; in addition, the double ratios of PSHr/TRPS and PSHt/PSHt + PDS were also calculated for E/S. The mean E/S values for PSHr and PSHt were significantly different for all types of lesion compared with control samples. The E/S ratios for apparently normal tissue obtained from cervices with CIN or carcinoma were also significantly different compared with corresponding control values, indicating a field effect. There was a considerable degree of overlap between individual values in the control groups versus those obtained with each type of lesion. The corresponding mean E/S values for TRPS and for PSHt + PDS in the samples containing lesions were not significantly different from control means except for the group containing CaCx. However, the mean values for the double ratios (PSHr/TRPS and PSHt/PSHt + PDS) were significantly different in the groups containing lesions compared with the controls. Moreover, apparently normal tissue obtained from cervices containing CIN or carcinoma had different mean values compared with the controls, confirming the existence of a field effect. The degree of overlap of individual values in the lesion groups compared with the control values was much less with double ratio values than previously noted for single ratio values. In consequence, the double ratio measurements clearly discriminated CIN I + II and CIN-III from controls. Our data show that CIN is associated with marked changes in tissue protein thiols and disulfides and that these differences extend to neighboring apparently normal tissue indicative of a field effect.

Quantification of the histochemical staining for carbonyles and DNA using 3-hydroxy-2-naphthoic acid hydrazide and fast blue B.

Fixed cells and tissues pretreated with 4-hydroxynonenal were used as models for the histochemical demonstration of protein bound aldehydic groups. The aldehydes were stained with both a modification of the 2,4-dinitrophenylhydrazine method (2,4-DNPH) and the optimized staining using 3-hydroxy-2-naphthoic acid hydrazide and Fast blue B (NAH-FB). A correlation has been found between the specific microphotometric mean integrated maximum absorbance values of cells and tissues stained with 2,4-DNPH and with NAH-FB (cc = 0.999). The maximum absorbance measured after 2,4-DNPH-staining (epsilon 367 = 21,000) were 1.893 +/- 0.072 (P less than 0.01) times that of NAH-FB-staining at 550 nm. Microphotometrically determined DNA-values of different cells stained with the NAH-FB-DNA-method correlated with those determined with methods of analytical biochemistry and published by other authors.

Histophotometry of protein thiols and disulphides in tissue samples from the human uterine cervix and the skin reveals a "field effect" as well as an "extended field effect" of malignant tumours.

Fresh frozen and fixed serial sections were stained with 2,2'-dihydroxy-6,6'-dinaphthyldisulfide (DDD) and Fast blue B for reactive protein thiols (PSHr) and total reactive protein sulfur (TRPS). The mean optical densities of PSHr and TRPS determined histophotometrically at a distinct part of a tissue were related to each other (PSHr: TRPS). If this quotient has been determined, e.g. for normal epithelium and the adjacent stroma, both quotients can be related to each other by a double quotient (Q PSHr: TRPS). With the aid of the double quotient highly significant differences could be found between tumours and normal tissue from patients without tumour of the human uterine cervix. Similar differences exist between normal skin from healthy patients and skin tumours. Q PSHr: TRPS revealed similar differences to exist between normal tissue of patients without tumour and apparently normal tissue in the neighbourhood of tumours of the uterine cervix and of skin ("field effect" of tumours). Histophotometric investigations on abdominal skin (and skin of breast) showed highly significant differences between normal skin of patients without tumour and patients with various kinds of tumours of the uterine cervix, ovaries, liver and breast ("extended field effect" of tumours).

Optimization of the histochemical demonstration of DNA using 3-hydroxy-2-naphthoic acid hydrazide and fast blue B.

Previous methods for the histochemical demonstration of DNA were optimized. p-Toluene sulfonic acid as catalyst for hydrazone formation between the aldehydes generated after Feulgen hydrolysis and 3-hydroxy-2-naphthoic acid hydrazide (NAH) was used instead of acetic acid. Modifications of the conditions of the coupling reaction with Fast Blue B reduced the background staining. The optimized histochemical staining method for DNA (NAH-FB-DNA staining) can be performed easily and reproducibly. Without prior Feulgen hydrolysis the optimized method can also be used for the histochemical demonstration of reactive carbonyls undissolved under the given histochemical conditions.

Histophotometric quantification of the field effect and the extended field effect of tumors.

Histophotometric investigations have been made on samples of human skin. Fresh frozen serial sections were fixed and stained for either reactive protein thiols (PSHr) or total reactive protein sulphur (TRPS) using modifications of the DDD-Fast blue B-method. In addition, total protein thiols (PSHt) were stained with the Mercurochromcyanide-method, and proteins were stained using a modified amido-black procedure. Significant differences were found between the different tumours investigated and normal tissue, and also between apparently normal tissue adjacent to the tumours and normal tissue from patients without tumour. To reveal such tumour-related changes of apparently normal tissue, termed the field effect of tumours, a double quotient had to be calculated from the PSHr-and TRPS-values determined from both epithelium (epidermis) and connective tissue. In addition, abdominal skin was investigated from patients without tumour and patients with tumours of the female genital tract, liver or breast. With the aid of the double quotient procedure, highly significant differences were found between normal abdominal skin of patients without tumours versus similar samples taken from patients with tumours. The tumour-related changes found with abdominal skin distant from the tumours have been termed the extended field effect of tumours. These general tumour-related changes, independent of the size, state or degree of malignancy of the distant tumour, could be shown to be due to changes in abdominal dermis.

Histochemical demonstration of primary amino groups with 2-hydroxy-1-naphthaldehyde (HNA): optimization of the method.

2-hydroxy-1-naphthaldehyde (HNA) was used for the histochemical demonstration of primary amino groups. The Schiff-bases formed exhibited a double-banded maximum absorption at lambda = 420 nm and lambda = 400 nm, respectively. The dependence of the equilibrium of the Schiff-base formation upon the concentration of HNA, the reaction medium, especially upon its pH-value, was investigated with Ehrlich ascites tumour cells and rat liver parenchymal cells. Optimum conditions were elaborated for the histochemical reaction running either in absolute ethanol (HNA-EtOH) or in a mixture of ethanol and acetate buffer pH = 4 (HNA-pH4), the equilibrium of the reactions adjusted after 10 d and 1 d, respectively. The HNA-pH4-method can be used for the histochemical demonstration of primary alpha-, delta-, and epsilon-amino groups of proteins. The HNA-EtOH-method comprises primary amino groups of proteins and of other substances insoluble in absolute ethanol. Both histochemical methods proved to be reproducible.

Changes in protein sulfur groups in hepatocytes of newborn rats under glucocorticoid stimulation.

A daily administration of hydrocortisone-acetate (25 micrograms/g b.w.) increased both dry mass and protein content of the hepatocytes of newborn rats by 84% and 89% respectively, after 5-day-treatment. The increase correlated with the duration of hormonal treatment. As an average per cell, the total reactive protein sulfur increased up to 127%. This increase depends on a major increment of thiols (+179%) and on a minor increment of disulfides (+18%). Within the thiols, the fast reactive ones exhibited the most pronounced increment (+214%). Per protein unit, the total reactive sulfur increased, after a 1 day lag period, by up to +20%. Thiols showed a 47%-increment due to increase of fast reactive thiols (+70%) more than of slow reactive thiols (+20%). On the contrary, disulfides decreased (-37%). Consequently, the protein thiol/disulfide ratio shifted from 2.14 in control hepatocytes to 4.98 (+133%) in hormone-stimulated hepatocytes. Both the increase of the thiol content, and the shift of the SH/SS-equilibrium of the cellular proteins, correlated with a concomitant increase of enzymic activities such as gamma-glutamyltranspeptidase, glutathione reductase, glutathione peroxidase, glutathione S-transferase and 7-ethoxycoumarin O-deethylase.

Quantitative cytospectrophotometric studies on protein thiols and reactive protein disulphides in samples of normal human uterine cervix and on samples obtained from patients with dysplasia or carcinoma-in-situ.

Quantitative microspectophotometric studies have been made on sections of human cervix after staining for reactive protein thiol-groups (PSHr), and the sum of protein thiols with so-called reactive protein disulphides (together abbreviated as TRPS). Measurements were made on normal epithelium, apparently normal epithelium adjacent to a pathological lesion, dysplastic epithelium, carcinoma-in-situ, and adjoining stroma. The numbers of cases studied were: normal healthy controls (53); patients with dysplasias (34) and patients with carcinoma-in-situ (29). In the normal control sections the ratio of PSHr in epithelium:stroma was approximately 2.7 and this ratio was strongly decreased in dysplasias (1.6) and carcinoma-in-situ (1.5); the 3 populations of values had sufficient overlap to prevent this measurement being an effective discriminator. No significant variations were observed with TRPS-values except with changes in the stroma adjacent to apparently normal epithelium. However, the ratio of PSHr:TRPS was effectively discriminatory when this double-staining ratio was calculated for epithelial values:stromal values. These results are discussed in relation to the importance of thiol-groups in cell division and cancer, and the biological implications of similar changes observed in neighbouring apparently normal epithelium.

Histophotometric quantification of protein and reactive proteinthiols in invasive carcinomas of the skin.

Frozen sections cut from 14 samples of invasive carcinomas of the skin were stained with Amido black for protein determination and with dihydroxydinaphthyldisulphide fast blue to quantify reactive protein thiols (PSHr) and were then analysed microphotometrically. It was found that all of the samples exhibited significant reductions in protein levels (49%-74%) and PSHr levels (32%-53%) as compared to normal epidermis. Thus, the content of proteins of PSHr groups was 1.7 times greater in the malignant tissue examined than in normal epidermis. These results are in accordance with those previously obtained in basal-cell epitheliomas.

Quantitative microphotometric studies of Feulgen-stained nuclei on sections of the human uterine cervix: optimization of preparation and measuring technique.

The total extinction of apparently normal intermediate cell nuclei was microphotometrically measured in formalin-fixed Feulgen-stained paraffin sections of the uterine cervix. We investigated whether reproducible microphotometric results can be obtained from a histologic section expected to contain a certain amount of sectioned nuclei and stained by a standard technique. The results have shown that exact reproduction of microphotometrically measured mean total extinction values of intermediate cell nuclei can be achieved with a threshold value of 90% transmission in 10 microns sections.

Protein thiols in normal and neoplastic human uterine cervix.

Protein-thiol groups that react with dihydroxydinaphthyl disulphide during a 7 h incubation (so-called reactive protein thiols, PSHr) have been quantitatively measured on sections of human uterine cervix by microcytospectrophotometry. Measurements were made on areas (1 micron 2) of epithelium and adjoining stroma in samples of normal cervix, and in samples obtained from patients with dysplasia, carcinoma-in-situ and invasive cancer. The ratio of PSHr in epithelium to stroma is substantially reduced in the pathological conditions compared with normal and in apparently normal adjacent areas. Such changes in PSHr are discussed in relation to the redox balance of the tissue, and free radical disturbances previously described.

Significant decreases in the intensity of staining for proteins and protein thiols in basal-cell epitheliomas (basaliomas) as compared to normal skin.

Microphotometric measurements of fast reacting protein thiols (PSHr) and proteins were performed on freshly frozen sections of samples from normal skin (26 cases as controls) and from 45 basal cell epitheliomas (basalioma; BCE). The intensity of the staining (E/micron2) for both proteins and PSHr was significantly higher in normal epidermis than in the adjacent dermis. The values of QE (quotient of values observed in the epidermis divided by those observed in the dermis) were calculated to be 3.48 for proteins (QE, Prot) and 4.62 for PSHr (QE, PSHr). In cases of BCE, significantly lower QE values were found: QE, Prot = 2.16 and QE, PSHr = 1.72. The decrease of QE, PSHr was due to a decrease in the staining intensity observed in the BCEs, whereas practically no changes occurred in the adjacent dermis. The decrease of QE, Prot was mainly caused by a decrease in the staining intensity in the BCE (by 68%) as well as in the adjacent dermis (by 36%). By dividing the mean extinction value (E/micron2) for PSHr by the E/micron2 for proteins, a new quotient, PSHr/Prot, is obtained which can serve as a quantitative measure of the content of the tissue proteins of PSHr. The proteins of normal epidermis contained more PSHr than dermal proteins. The proteins of BCEs also contained more PSHr than those of the adjacent dermis, but the PSHr/Prot values of both tissues were 1.5 to 1.6 times greater than the corresponding values for normal epidermis and dermis, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

A modification of the amidoblack-TCA-staining for quantitative microspectrometrical determination of proteins in tissue sections.

Conditions are described by which cells and fresh frozen tissues, following fixation with ethanol-ether, and after staining with the amidoblack (AB)-TCA-staining method, show a modified dye/protein ratio of 0.9 moles AB/10(5) g protein compared to 8.5 moles AB/10(5) g protein ( Schauenstein et al. 1980) as in a previously used method. In contrast to the AB-TCA-method, which leads to extremly high and unmeasurable extinctions in tissue sections, staining with the modified AB-TCA- 23st -method with 10 microns tissue sections produces easily measurable extinction values. A correlation of the microspectrometrically determined mean total extinction values of different cell types and nuclei after staining with the tetrazonium method (N ohammer 1978; N ohammer and Desoye 1981) and on the other hand with the AB-TCA 23st -method has been found. The microspectrometrically determined extinctions after AB-TCA 23st -staining can be calculated; an extinction of 0.04248 corresponds to 1 pgm protein.

The microspectrometrical determination of the nucleus-total-cell-protein-ratio: a possibility of objective cell type analysis.

Cells from smears of the normal human squamous epithelium of the gingiva, fixed and stained for protein using the tetrazonium method optimized by Nöhammer and calibrated by Nöhammer et al., were investigated. The extinctions of both the total cells and of the rectangular areas circumscribing the nuclei, were measured microspectrometrically. Altogether 417 cells from 6 healthy persons of both sexes were investigated. 9 distinct subgroups of cells were found showing an exact linear correlation between nuclear and total cell extinctions. In the graph of both the nuclear and the total cell extinctions the 9 subgroups can be seen as 9 distinct linear groups of points, defined exactly by their regression lines. Thus, every squamous epithelial cell within the smear can be typed definitely and objectively in respect to its membership of one of these 9 linear groups of points. The obviously definite, legitimate connection between the extinctions of the total cells and of their nuclei affords a glimpse into the processes of cellular differentiation and allows the definition of the so-called stem cell in terms of protein content of the total cell and of the nucleus.