K-ras gene point mutations and p21ras immunostaining in human ovarian tumors.

It is well recognized that genetic alterations within oncogenes, tumor suppressor genes, DNA mismatch repair and excision repair genes contribute to tumorigenesis within the human ovary. This study was undertaken to screen for the existence of K-ras gene point mutations in paraffin-embedded slides randomly selected from benign and malignant ovarian tumors applying the PCR-RFLP technique. Expression of p21ras was also assessed in 30 primary ovarian adenocarcinomas immunohistochemically. K-ras codon 12 point mutations occurred in nine of 40 (22.5%) cases. They were not identified in two benign mucinous cystadenomas, but in one out of two (50%) mucinous tumors of LMP (low malignant potential), in five out of 30 (17%) ovarian adenocarcinomas, and in one case of adenocarcinoma metastatic to the ovary. K-ras activation was also detected in one out of four (25%) sex cord-stromal cell tumors (folliculoma), and in one dysgerminoma. None of these tumors exhibited K-ras codon 13 point mutations. Gene alterations were more frequently found in mucinous than in non-mucinous (30% vs 10%) tumors, although the difference did not reach significance (p > 0.05). The frequency of K-ras point mutations was correlated neither with clinical nor with pathological variables of cancer. Cytoplasmic p21ras was expressed in all adenocarcinomas negative for K-ras point mutations, whereas one of five (20%) K-ras-positive tumors exhibited lack of immunoreactivity. In conclusion, these findings confirm the role of K-ras activation in mucinous ovarian tumors. p21ras expression is not necessarily associated with K-ras gene alterations in human ovarian adenocarcinomas.

Evaluation of DNA mismatch repair system in cervical dysplasias and invasive carcinomas related to HPV infection.

UNLABELLED: The aim of this study was to answer the question whether the products of hMSH2 and hMLH1 genes take part in the mutation track of cervical carcinoma. METHODS: IgG1 monoclonal antibodies (Pharmingen) detecting epitopes characteristic of hMLH1 and hMSH2 were used in the present study. The value of the half-quantitative H-score coefficient was calculated. Its threshold value was 0.4. Identification of 16 and 18 HPV types was performed by PCR. RESULTS: An intensified hMLH1 protein expression was observed both in the squamous epithelial carcinomas and cervical adenocarcinomas (H-score of 1.44 and 0.98, respectively) as compared to the control (H-score of 0.9). However, a decreased expression of hMSH2 protein was observed in the analysed cases of carcinoma (0.9 and 0.7) as compared to the control group (1.2). An intensified expression in G3 for hMLH1 and higher hMLH1 in comparison to hMSH2 was observed. CONCLUSIONS: 1. A considerable expression of hMLH1 and hMLH1 proteins was observed in the tissues with invasive cervical carcinoma not only within epithelial but also in stromal cells. 2. More intense expression of hMLH1 and hMSH2 was observed in invasive carcinomas and CIN than in the non-neoplastic cervical tissue lesions (erosion). 3. A stronger expression was observed for the hMLH1 than for the hMSH2 proteins--contrary to the cases of carcinomas of the uterine corpus and endometrial carcinoma.

[Hydrophobic DNA adducts in relationship to estrogen and progesterone receptors content in human uterine cancer]. / Hydrofobowe addukty DNA w relacji do gestosci receptorów estrogenowych i progesteronowego w gruczolakorakach blony sluzowej macicy u kobiet.

OBJECTIVE: Determination of the relationship between hydrophobic DNA adducts (A) and estrogen receptors (ER) and progesterone (PR) receptor status in uterine cancers. METHODS: Using the P1 enriched version of 32P-postlabeling for hydrophobic DNA adducts detection on polyethyleneimine (PEI) cellulose thin layer chromatograms (TLC) we examined 11 uterine cancer DNAs. The quantification of the adducts was performed by Cerenkov counting of the spots. ER and PR status was recognized histochemically and H-score estimate was performed for each investigated cancer tissue. Patterns of uterine cancer DNA adducts were compared to the maps of adducts recognized in normal human endometrium. RESULTS: In three of the studied uterine cancers there was no positive staining of ER and PR; in one case there was a weak ER staining but PR staining was negative. In ER negative tumors the A level was significantly higher than in ER positive cancers (138.1 +/- 64.1 vs. 49.7 +/- 26.8 adducts per 10(9) nucleotides, respectively, p < 0.05). Highest A levels were found in two ER and PR negative G3 metastatic tumors. Finally, in all investigated cancers there was a strong, inverse correlation between ER content and A level (r = -0.67, p < 0.03). In addition, the correlation between PR level and A was of borderline significance (r = -0.6, p = 0.053). The TLC patterns of adducts in uterine tumors were found to be qualitatively similar, but not quantitatively, to those observed in normal human endometrium DNA. CONCLUSION: The data presented suggest that the hydrophobic DNA adducts could play a role in a sex-steroid hormone independence of human endometrial cancers. The highest accumulation of DNA adducts was recognized in neoplasms displaying the most malignant phenotype.

Total sialic acid content in endometrial cancer tissue in relation to normal and hyperplastic human endometrium.

The aim of this study was to measure the total sialic acid (TSA) content in endometrial cancer tissue and to assess its relationship to clinicopathologic features of the malignancy. Tissue TSA content was measured in 42 women with endometrial cancer, 14 women with endometrial hyperplasia, and 45 women with normal endometrium in the proliferative phase (n = 16) and secretory phase (n = 29) of the menstrual cycle using the Warren procedure. The mean TSA content in endometrial cancer (2.16 micromol/gm) was significantly higher in comparison to normal endometrium in both proliferative (1.23 micromol/gm) and secretory (1.51 micromol/gm) phases. TSA content in the hyperplastic endometrium (1.56 micromol/gm) was higher as compared to the normal endometrium, but the differences were not statistically significant. An increased TSA content in the neoplastic endometrium in relation to the normal endometrium supports the view that the development of endometrial cancer is associated with the increasing content of sialic acid in the tumor tissue.

[Prognostic value of serum MMP-2 level in uterine cancer affected women]. / Prognostyczne znaczenie poziomu MMP-2 w surowicy krwi w przypadku gruczolakoraka blony sluzowej macicy u kobiet.

OBJECTIVES: Matrix metalloproteinase-2 (MMP-2) which can degrade type IV collagen is implicated in cancer invasion and metastasis. The aim of the study was to evaluate the serum MMP-2 level in patients with endometrial carcinoma (EC) and to compare the level with histological grade G1 to G3 in relation to clinical staging I degree-IV degrees as well as myometrial invasion (M. < 1/2, M. > 1/2). MATERIALS AND METHODS: The study group consisted of 30 patients with EC. MMP-2 serum levels were measured with specific one-step sandwich enzyme immunoassay (Amersham Life Science). Statistical analysis was performed by Mann-Whitney test and p value < 0.05 was considered as statistically significant. RESULTS: The statistically elevated enzyme level was observed in patients presenting II degrees-IV degrees clinical stage of EC in comparison to women with I degree stage of this carcinoma. There were no significant correlations between MMP-2 serum levels and grades of histological differentiation as well as depth of myometrium invasion. CONCLUSIONS: MMP-2 serum level is statistically higher in clinically advanced stages of EC. This enzyme seems to be useful in monitoring of therapeutical efficacy or screening for the recurrences of the disease.

[Overall genomic DNA methylation in relation to estrogen]. / Metylacja genomowego DNA w relacji do gestosci receptora estrogenowego i progesteronowego w gruczolakorakach blony sluzowej macicy u kobiet.

Overall genomic DNA methylation was analyzed using enzymatic digestion into nucleotides, 32P postlabeling, two-dimensional thin-layer chromatography on cellulose plates and phosphobioimaging quantitation, in relation to immunohistochemically measured estrogen (ER) and progesterone receptor (PR) status of 15 uterine cancers. Mean 5-methyldeoxycytosine (m5dC) content did not differ between ER-positive and ER-negative neoplasms. Highest values of m5dC were noted both in ER-negative and ER-positive tumors. Additionally, there was no low DNA methylation in ER negative uterine cancer tissues. Decrease of the overall genomic DNA methylation could be related to the increase of ER/PR ratio, however it was not significant in our investigation. The potential role of steroid receptors status in uterine cancer tissue is discussed.

Epidermal growth factor receptor immunostaining and epidermal growth factor receptor-tyrosine kinase activity in proliferative and neoplastic human endometrium.

Epidermal growth factor receptor (EGFR) expression and EGFR-tyrosine kinase (EGFR-TK) activity were measured in proliferative (n = 12) and neoplastic (n = 31) human endometrium. Immunoreactivity of EGFR was related to clinicopathological features, estrogen receptor (ER) and progesterone receptor (PR) status, and patient outcome. All proliferative and 27 neoplastic specimens expressed the EGFR. Expression of the EGFR was higher in proliferative endometrium than in endometrial cancer (p < 0.0001). ER immunostaining was observed in 19 of the endometrial carcinomas, while PR expression was demonstrated in only 12 neoplastic specimens. EGFR expression was not related to the ER/PR immunostaining in endometrial carcinomas. Clinicopathological features (age, stage, histological type, grade or depth of invasion) and clinical outcome were unrelated to EGFR immunoreactivity. EGFR-TK activity was detected in 29 of 31 endometrial neoplasms with a 9 times higher mean activity in neoplastic than in proliferative endometrial specimens. There was no relationship between the EGFR-TK activity and EGFR immunostaining in human neoplastic endometrium (p = 0.77). A trend towards a poorer outcome of patients with the EGFR-TK activity above 40 pmol/min/mg was observed, but was not statistically significant. These results support the view that the EGFR expression is downregulated in endometrial carcinomas compared to proliferative endometrial specimens.

Evaluation of matrix metalloproteinases-1 and -3 concentrations in the tunica albuginea, the apical wall of atretic follicles and the corpus luteum of normal human ovaries.

Matrix metalloproteinases-1 (MMP-1) and -3 (MMP-3) are proteolytic enzymes involved in remodeling the ovarian extracellular matrix throughout the menstrual cycle. The aim of the present study was to evaluate the tissue concentrations of MMP-1 and MMP-3 in the apical wall of atretic follicles (androstenedione/estradiol ratio > 4), tunica albuginea dissected from the ovarian surface overlying areas devoid of follicles, corpus luteum, and tunica albuginea covering the corpus luteum. After extraction of MMPs from the tissue samples, their concentrations in the extracts were measured by enzyme-linked immunosorbent assays (ELISA). Significantly less MMP-1 was detected in the apical wall of atretic follicles compared to tunica albuginea taken from sites devoid of follicles. These data indicate that atresia is associated with relatively low concentrations of MMP-1 in the apical wall of the follicle. Moreover, there was a negative correlation between the amount of MMP-3 and the diameter of follicle. These data suggest that both MMPs play an important role in the final step of atresia. The amount of MMP-1 in the corpus luteum was several times lower than in the other tissues. This is likely due to stabilization of the extracellular matrix during the period of the corpus luteum maintenance. The concentration of MMP-3 did not differ significantly among the examined tissues.

Interstitial collagenase (MMP-1) activity in human ovarian tissue.

The collagen content and collagenase activity were estimated in human ovarian interstitial tissue devoid of all visible follicles in menstruating, fertile as well as climacteric women. The mean total collagenase activity in ovarian specimens taken during both follicular (n = 10, 3.97 +/- 0.58 U/g wet weight, ww) and luteal phase (n = 10, 3.39 +/- 1.24 U/g ww) of the normal menstrual cycle along with total collagen concentration (184.8 +/- 41.0 vs. 194.4 +/- 30.5 micrograms/mg ww, respectively) did not differ. Total collagenase activity of climacteric gonads (n = 5, 1.55 +/- 0.71 U/g ww) was lower than in specimens collected during both follicular and luteal phase (p = 0.0002 and p = 0.017, respectively). About 23% of the total collagenase activity in follicular phase ovarian extracts and only about 1% in luteal phase ovarian preparations was found in the latent form. The percentage of latent collagenase in ovarian tissue during the follicular phase was negatively correlated with the day of the menstrual cycle (r = -0.93, p = 0.007). Extracellular matrix remodelling in the human ovary can be correlated with the functional status of the follicular unit.

Serum leptin concentrations in women taking oral contraceptives.

OBJECTIVES: To investigate serum leptin concentrations in women taking oral contraceptives containing the same gestagen and different doses of ethinyl estradiol. STUDY DESIGN: 30 women received tablets containing 20 microg of ethinyl estradiol (EE) and 150 microg of desogestrel (DSG) (Mercilon) whereas another group of 30 women received 30 microg of EE and 150 microg of DSG (Marvelon). Serum leptin concentrations were estimated using a Leptin RIA kit (Linco Research USA) after an overnight fast on the first day of the cycle prior to the onset of therapy as well as after the 3rd and 6th treated cycles. RESULTS: In both groups a positive correlation between serum leptin and body mass index (BMI) was found (r=0.56; P<0.001 and r=0.67; P<0.001). The initial serum leptin concentration in the Mercilon group was 7.62+/-8.46 ng/ml. This value was not statistically different from values after 3 months (9.31 8.23 ng/ml) and after 6 months (10.53+/-8.03 ng/ml) of treatment. Very similar results were found in patients receiving Marvelon: 8.81+/-6.56 ng/ml initially; 11.62+/-11.16 ng/ml at 3 months, and 10.38+/-7.32 ng/ml at 6 months. The statistical analysis did not reveal any significant difference at each investigated time point in either study group. CONCLUSIONS: Modern low dose OC containing third generation gestagen and low dose of ethinyl estradiol, does not have any influence on serum leptin or BMI, and therefore does not exert a significant influence on body energy metabolism.

Concentration of an epidermal growth factor in blood serum of males during topical treatment of psoriasis.

Epidermal growth factor (EGF) is a mitogen that stimulates cell division of various cells of epidermal origin. The present study was undertaken to clarify whether the serum level of EGF is correlated with the disease activity during local therapy with dithranol in psoriasis. We examined serum EGF concentrations in acute and chronic psoriasis before and after topical treatment with dithranol and the correlation with Psoriasis Activity and Severity Index (PASI). Male patients were divided into two groups: acute psoriasis (AP, 18 cases) and chronic psoriasis (CP, 17 cases). A control group C consisted of 20 healthy male volunteers. Radioimmunoassay of EGF was performed using the reagent pack (Amersham, UK). In the CP group mean EGF was higher before treatment than in the AP and C groups, but not significantly. EGF concentration after local treatment was higher in the CP group than the AP group (P < 0.02); the AP group, however, showed statistically significant decrease of EGF after the treatment (P < 0.04). No correlation between EGF and PASI was found. Serum EGF concentration increased in 19/35 treated patients.

Role of fascial collagen in stress urinary incontinence.

OBJECTIVES: Our purpose was to determine whether collagen of the pubocervical fasciae that support the urethrovesical junction undergoes alterations that might contribute to incontinence. STUDY DESIGN: Pubocervical fascia was collected as a residual tissue in 82 patients, aged 25 to 73 years, during surgical treatment of cystocele (n = 26, no incontinence) or of stress urinary incontinence (n = 56). Measurements were made of collagen content, solubility, and cross-linking and of collagenase activity. RESULTS: Patients treated for incontinence had the same mean age and parity as the control cystocele group. There was a highly significant (20%, P <.0005) decrease in collagen content in fascial tissue from incontinent women. There was no difference in the percentage of acid-soluble (0.7%) and pepsin-soluble (17%) collagen in the 2 groups of patients; this agrees with the lack of significant change in the degree of collagen cross-linking by pyridinoline. Collagenase activity was significant in fascia but did not change in incontinence. Incontinent women had an increased body mass index. CONCLUSIONS: The pubocervical fasciae of incontinent women show a diminished content of collagen, but this is not accompanied by changes in collagen solubility or cross-linking or in collagenase activity. This decrease in collagen may contribute to the weakening of support of the bladder neck.

Immunohistochemical expression of syndecan-1 in human endometrial cancer cells.

There are indications of increased frequency of endometrial cancer, one of the most common malignancies in women. Tissue samples of normal and malignant endometria were obtained post operatively from 30 women. We noted expression of syndecan-1 in 40% of investigated cancers. The most differentiated cancers showed 75% of positively stained specimens, moderately differentiated 40% and poorly differentiated neoplasm did not stain at all. In normal endometrial tissue syndecan-1 expression was regular and distinct in each specimen, but immunoreactivity of the hyperplastic endometrial specimens was absent. The detection of syndecan-1 in endometrial cancer of different clinical and histological stages could be of prognostic value in clinical diagnosis.

Local collagen turnover in human foetal membranes during full term vaginal delivery.

OBJECTIVES: To investigate if changes in local metabolism of collagens type I and type III may contribute to the term rupture of the foetal membranes. STUDY DESIGN: In extracts from membranes taken along the rupture line and near the site of the umbilical cord attachment to the placenta, we measured concentrations of specific markers of collagen synthesis, namely C-terminal propeptide of type I collagen (PICP) and N-terminal propeptide of type III collagen (PIIINP), as well as the concentration of the pyridinoline cross-linked C-terminal telopeptide of type I collagen (ICTP), which reflects the rate of collagen type I breakdown. RESULTS: The mean concentrations of PICP and PIIINP did not differ statistically between the two examined groups of samples. The mean ICTP concentration was threefold higher in extracts prepared from samples taken near the rupture site, than from membranes derived from the proximity of the umbilical cord attachment. CONCLUSIONS: Term rupture of foetal membranes is accompanied by increased local degradation of type I collagen fibrils at the rupture site, whereas the biosynthesis of collagen types I and III remains unchanged.

[The analysis of serum lipid profile in anovulatory and ovulatory women in fifth decade of life]. / Analiza profilu lipidowego u kobiet w piatej dekadzie zycia w cyklach owulacyjnych i bezowulacyjnych.

OBJECTIVES: We examined the effect of declining ovarian function on lipid profile. MATERIALS AND METHODS: Serum concentrations of total cholesterol (TC), low density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol (HDL), triglycerides (TG) as well as early follicular phase FSH, periovulatory 17-beta oestradiol and midluteal progesterone levels were estimated. 71 regularly menstruating premenopausal women were divided according to serum progesterone concentration on: anovulatory (progesterone < 4.5 ng/ml) and ovulatory group (progesterone > 4.5 ng/ml). RESULTS: Investigated groups did not differ with respect to mean age, body mass index but hormonal profile showed higher level of FSH in anovulatory (15.1 +/- 21.2 mIU/ml) then in ovulatory group (5.1 +/- 4.2 mIU/ml, p = 0.008). 17-beta oestradiol levels were 91.9 +/- 29.9 pg/ml vs. 123 +/- 48.6 pg/ml, p < 0.05, respectively. Statistically significant difference in LDL concentration between anovulatory and ovulatory group was found (144.9 +/- 29.9 mg/dl vs. 131.4 +/- 24.6 mg/dl, p = 0.04, respectively). Also HDL content of the TC showed statistically significant difference between these two groups (19.3 +/- 4.4% vs. 21.4 +/- 4.3%, p = 0.04, respectively). However, there were no difference in serum concentrations of TC, TG, HDL. CONCLUSION: Based on these findings we concluded that changes in lipid metabolism that lead to increased risk of cardiovascular diseases may appear as early as in the 5th decade of life in apparently healthy women.

[Activity of tissue inhibitors of metal proteinases (TMI) in th fetal membrane during term vaginal delivery]. / Aktywnosc tkankowych inhibitorów metaloproteinaz (TIMPs) w blonach plodowych podczas porodu w ciazy donoszonej.

In the foetal membranes that ruptured spontaneously or by means of amniotomy during term vaginal deliveries the activity of tissue inhibitors of metalloproteinases was evaluated. Samples were taken from the proximity of the rupture line. Almost twofold lower TIMPs activity in the naturally ruptured membranes was observed. We conclude that during normally proceeding term vaginal delivery the activity of TIMPs in the lowermost pole of the amniotic sac is decreased, what may play very important role in the mechanism of the term rupture of the foetal membranes.

Local activity of matrix metalloproteinases in a case of botryoid sarcoma.

The aim of the study was to assess the activities of the collagenases type IV (matrix metalloproteinase type 2 [MMP-2] and matrix metalloproteinase type 9 [MMP-9]), also known as gelatinases, and the local activity of interstitial collagenase (matrix metalloproteinase type I[MMP-1]) in tissue extracts from a case of the botryoid sarcoma, a rare and very malignant tumour of the female genital tract. Zymography revealed that botryoid sarcoma does not express the 92-kDa form of type IV collagenase activity in Triton extract and only weak activity in Heat extract when compared to values found in extracts from striated muscle and fibroma uteri. MMP-1 appeared in the latent form only in the Triton extract of botryoid sarcoma and its activity was lower than those found in the control tissues. These results indicate that the very rapid local invasion and systemic metastases associated with botryoid sarcoma do not depend on the activity of tumour-derived gelatinases.

Extracellular matrix remodelling within the normal human ovarian capsule.

Collagen tissue content and both interstitial (MMP-1) and type IV collagenases (also known as gelatinases) activity within the normal human ovarian capsule were investigated. The apical tunica albuginea (n = 10) displayed a lower mean total collagen concentration than the ovarian capsule areas (n = 9) with no follicles underneath them (137.8 +/- 36.1 vs. 176.6 +/- 23.1 micrograms/mg wet weight tissue (ww), P = 0.004). This was accompanied by higher net interstitial collagenase activity (12.96 +/- 2.26 vs. 5.97 +/- 1.9 U/g ww, P = 0.016) which was present within the ovarian capsule in active form only. Zymography revealed the dominance of the 72-kDa over the 92-kDa gelatinase form regardless of the capsule area investigated. Our results indicate that extracellular matrix remodelling within human tunica albuginea is more strongly pronounced in the apical region.

[Tissue levels of EGF and collagen in ovaries of women]. / Tkankowe stezenia EGF i kolagenu w jajnikach u kobiet.

Tissue EGF concentration as well as collagen content were investigated in ovarian homogenates from both menstruating and menopausal women. EGF concentration was found to be higher, but not significant, in postmenopausal women, whereas total collagen as well as forms soluble in acetic acid and in this acid with pepsin were slightly higher in women with normal menstrual cycle. There were statistically significant negative correlations between above mentioned forms of collagen as well as ovarian EGF concentration in this group of patients. The potential role of epidermal growth factor in modulation of ovarian function and extracellular matrix remodelling within human ovary is discussed.

[Biochemical changes in extracellular matrix components of the amniotic sac during its premature rupture]. / Zmiany biochemiczne macierzy zewnatrzkomórkowej blon plodowych przy ich przedwczesnym pekaniu.

In the fetal membranes that have ruptured 12 hours before onset of the labour the tissue collagen content was lower than in control accompanying by similar local collagenase and elastase activity. These results excluded the inflammatory process as the main cause of prelabour preterm rupture of membranes (PPROM). The analysis of the collagen fractions taken from the site of rupture and the location close to umbilical cord indicates that in PPROM there are changes in posttranslational collagen molecule modification.