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HIF-1alpha induces GLUT-1 expression, and their presence has been evaluated in colorectal cancer. However, the expressions of GLUT-1 and HIF-1alpha have not been investigated together with reference to clinicopathological characteristics in human colorectal cancer. The aim of our study was to compare the expression of HIF-1alpha and GLUT-1 with various clinicopathological features of colorectal cancer. The presence of HIF-1alpha and GLUT-1 was visualized immunohistochemically in 123 primary tumors. Membranous localization of GLUT-1 was found in multifocally necrotizing cancer samples, while pure cytoplasmic perinuclear, mostly supranuclear GLUT-1 accumulation was characteristic of cancer fields with lack of necrosis. HIF-1alpha was located in the cytoplasm and occasionally in the nuclei of cancer cells. Immunoreactivity to GLUT-1 was significantly higher in node-positive cancers compared with nodenegative ones (p=0.04), confirming our earlier results obtained on a larger number of patients. Non-mucinous adenocarcinomas expressed GLUT-1 and HIF-1alpha with significantly greater frequency than mucinous adenocarcinomas (p=0.002, p=0.0002, respectively). GLUT-1 and HIF-1alpha expression did not differ in relation to tumor stage, location, or patients' age or gender. In contrast to that of GLUT-1, expression of HIF-1alpha correlated with grade (p=0.00003) without difference with regard to pN status. HIF-1alpha expression correlated with GLUT-1 expression in the whole patient population, as well as in all clinicopathological groups except for the pT1+pT2 group. Although the coexpression of cytoplasmic HIF-1alpha and GLUT-1 does not directly prove the dependence between HIF-1 as a nuclear transcriptional factor and GLUT-1 as its downstream protein, it is evidence of their simultaneous upregulation. The extranuclear accumulation of HIF-1alpha and GLUT-1 requires further studies to explain its significance in colorectal cancer.

Polyunsaturated free fatty acids (PUFAs) participate in normal functioning of the cell, particularly in control intracellular cell signalling. As nutritional components they compose a human diet with an indirect promoting influence on tumourogenesis. The PUFAs level depends on the functional state of the membrane. This work is focused on changes only of free unsaturated fatty acids amount (AA - arachidonic acid, LA - linoleic acid, ALA - alpha-linolenic acid, palmitoleic acid (PA) and oleic acid) in cell membranes of colorectal cancer of pT3 stage, G2 grade without metastasis. Qualitative and quantitative composition of free unsaturated fatty acids in the membrane was determined by high-performance liquid chromatography. It was shown that the malignant transformation was accompanied by a decrease in amount of LA and ALA while arachidonic and oleic acids increased. It is of interest that free AA levels are elevated in colon cancer, as AA is the precursor to biologically active eicosanoids.

Leptin and its receptor may be engaged in pathogenesis of breast cancer among various human tumors. In vitro investigations showed leptin-mediated escalation of estrogen synthesis and boosted activity of estrogen receptor ERalpha. Furthermore, leptin induced growth of malignant cells, counteracted apoptosis and stimulated cell migration as well as overexpression of angiogenic factors and degrading enzymes that split network of intercellular matrix. On the other side, leptin has been reported to favor apoptosis, lately. Proapoptotic effect of leptin action was revealed in interstitial cells of bone marrow and adipocytes. Our past reports provide evidences for overexpression of leptin and its receptor in breast cancer in comparison with benign mammary lesions. In current study we aimed at assessment of eventual relationships between leptin, leptin receptor and selected protein regulators of apoptosis in breast cancer. We applied immunohistochemistry for leptin, leptin receptor, anti-apoptotic Bcl-2 and Bcl-xL as well as pro-apoptotic Bak and Bax expression assessment in 106 cases of human breast cancers. The immunoreaction was graded and statistically evaluated. Expression of leptin was positively correlated with Bcl-xL, Bak and Bax (p<0.001, r=0.614; p<0.001, r=0.518; p<0.001, r=0.511, respectively). Statistical significances were noted between expression of leptin receptor and Bcl-xL or Bax (p=0.011, r=0.210; p<0.001, r=0.313, respectively). No correlation was encountered between leptin and Bcl-2, either leptin receptor and Bcl-2 or leptin receptor and Bak. On the basis of obtained results, leptin system could interfere in balance among expressions of pro- and anti-apoptotic proteins and regulate cell turnover and--by means of it--facilitate breast cancer progression.

Gap junctions are specialized cell membrane channels composed of connexins (Cxs), which mediate the direct passage of small molecules between adjacent cells. They are involved in the regulation of cell cycle, cell signaling and differentiation as well as probably invasion and metastasis. Up to now, Cx32 status in human breast cancer has not been studied. Consequently, the aim of the present study was the evaluation of the expression of connexin 32 (Cx32) in primary breast tumors (PTs) and matched-paired metastases to lymph nodes (MLNs) in correlation with selected clinicopathological features. Tissue samples from 79 women were examined by immunohistochemistry, using the streptavidin-biotin-peroxidase complex technique for Cx32. Cytoplasmic expression of Cx32 was detected in 31 of 79 breast cancers (39.2%). Both epithelial and myoepithelial cells of normal ducts adjacent to the tumor did not express Cx32. Increased expression of studied Cx was observed in metastases to lymph nodes relative to primary tumors. Additionally, Cx32-negative primary tumors developed Cx32-positive metastases. Statistical comparisons of Cx32 expression in the matched pairs indicate that this protein significantly increased in lymph node metastases compared to primary tumors (p<0.001). The expression of Cx32 in primary breast cancer was not statistically associated with age of patients, tumor size, lymph node status, but we observed a tendency toward association between Cx32 expression and histological differentiation. In conclusion, transformed cells may have an ability to produce Cxs also atypical for normal cells. Increased expression of Cx32 in metastases to the lymph nodes might reflect alteration in connexin gene transcription during breast carcinogenesis and finally, it may be a sign of more malignant phenotype of cancerous cells.

Aims and Background: Erythropoietin, VEGF, VE-cadherin are involved in angiogenesis. Besides that erythropoietin stimulates erythropoiesis and increases haemoglobin and hematocrit levels as well. Moreover, erythropoietin could directly stimulate colorectal cancer cell growth due to the presence of both erythropoietin receptor and erythropoietin production in malignant cells of this neoplasm. Therefore we aimed at measurement and comparison of serum erythropoietin with VEGF, VE-cadherin levels, blood haemoglobin and hematocrit in colorectal cancer patients of different clinicopathological profiles., Methods: We applied ELISA kits to evaluate preoperative serum levels of endogenous erythropoietin, VEGF and VE-cadherin in samples from 92 colorectal cancer patients and control group of 16 healthy volunteers., Results: Endogenous erythropoietin was significantly elevated in preoperative sera in colorectal cancer patients (p = 0.013) compared with healthy volunteers, however, erythropoietin levels were not significantly higher with the advancement of colorectal cancer. There were significantly higher levels of erythropoietin in the group of anaemic men in comparison to men with normal haemoglobin levels (p < 0.0001). VEGF and VE-cadherin did not correlate with erythropoietin. Erythropoietin levels negatively correlated with haemoglobin and hematocrit levels in all cancer patients; particularly in node positive cancers (N+), moderately differentiated tumours (G2) and deeply invading neoplasms (pT3+pT4)., Conclusions: Erythropoietin levels increase in colorectal cancer but circulating erythropoietin does not associate with progression of the disease. Thus, the use of recombinant erythropoietin seems to be safe. Our results suggest that negative feedback regulation persists between haemoglobin and erythropoietin in colorectal cancer. Production of erythropoietin remains therefore anaemia-associated, hypoxia-dependent and doesn't seem to be autonomic despite abundant expression of erythropoietin by colorectal cancers.

Recent studies suggested that Ob (Ob) and its receptor (ObR) could be involved in the pathogenesis of various human malignancies, among others in endometrial cancer. Moreover, hypoxia, which is associated with solid tumors, might stimulate, through hypoxia-inducible factor 1alpha (HIF-1alpha), expression of Ob and ObR. In this article, we analyzed by immunohistochemistry the expression of Ob, ObR, and HIF-1alpha in 60 cases of human endometrial cancer tissues as well as in 25 cases of normal endometria. Additionally, we assessed correlations among studied proteins as well as relationships with selected clinicopathological features of endometrial cancer. Immunoreactivity for Ob, ObR, and HIF-1alpha protein was observed in 56.7%, 30.0%, and 78.3% of endometrial cancers, respectively. The expression of HIF-1alpha showed a significant positive correlation with Ob (P < 0.0001, r = 0.573) and ObR (P = 0.020, r = 0.299). Moreover, we noted positive correlation between Ob and ObR (P = 0.001, r = 0.429). No statistically significant relationship was revealed between Ob, ObR, and HIF-1alpha protein in regard to patient's age, histological grade, and extent of tumor growth (pT). In conclusion, HIF-1alpha, which is related to tissue hypoxia in endometrial cancer, seems to be associated with overexpression of Ob and ObR. Ob could exert autocrine effect to stimulate endometrial cancer progression. Thus the autocrine Ob loop should be taken into consideration as a novel potential target in endometrial cancer prevention and treatment.

Unlabelled: Colon cancer morbidity in Poland is still increasing. During the course of the disease thromboembolic complications are often observed. It was documented that components of the hemostatic system influence cancer progression. The presence of coagulation factors was revealed in colon cancer tissue. So far the information on the presence of inhibitors of coagulation in this tumor type was lacking. The aim of the study was to evaluate the expression of coagulation inhibitors in loco in colon cancer., Material and Methods: Immunohistochemical method employing polyclonal antibodies directed against tissue factor pathway inhibitor (TFPI), antithrombin III (AT Ill), protein C (PC) and protein S (PS) was introduced., Results: The presence of TFPI was observed in cancer cells in a part of examined specimens, but the intensity of the staining was different. In most cases of colon cancer AT III expression of low intensity was revealed in a few cancer foci. In approximately 15% of cases there was no AT Ill expression, while in other 15% of examined fragments of colon cancer AT Ill was readily detected. Weak expression of TM was observed in most colon cancer tissues, but only in some cancer foci. In 1/3 of examined cases the strong expression of TM was revealed. Weak and heterogeneous intensity of expression of PC was demonstrated in about 75% of colon cancer cases, whereas in about 25% of the specimens--the expression of this protein was strong. Furthermore in most cases there was no or weak expression of PS. Only in about 25% of the cases the staining for PS was strong. Expression of TFPI, AT III and PC was revealed in macrophages, whereas TFPI and AT Ill were detected in mucus in the lumen of neoplastic glands, and TM--in endothelial cells., Conclusions: Weak representation or the lack of coagulation inhibitors in colon cancer in most cases may facilitate coagulation activation in the tumor burden, and consequently promote colon cancer progression. Heterogeneous and inconstant presence of blood coagulation inhibitors in colon cancer in loco may suggest, that nonocoagulant biological activities of these inhibitors may influence the cancer growth or its inhibition in an individual patient. Obtained results suggest that introducing various forms of blood coagulation inhibitors in the treatment of colon cancer patients may be beneficial in selected group of patients.

Purpose: Recent in vitro studies suggested that the autocrine leptin loop might contribute to breast cancer development by enhancing cell growth and survival. To evaluate whether the leptin system could become a target in breast cancer therapy, we examined the expression of leptin and its receptor (ObR) in primary and metastatic breast cancer and noncancer mammary epithelium. We also studied whether the expression of leptin/ObR in breast cancer can be induced by obesity-related stimuli, such as elevated levels of insulin, insulin-like growth factor-I (IGF-I), estradiol, or hypoxic conditions., Experimental Design: The expression of leptin and ObR was examined by immunohistochemistry in 148 primary breast cancers and 66 breast cancer metastases as well as in 90 benign mammary lesions. The effects of insulin, IGF-I, estradiol, and hypoxia on leptin and ObR mRNA expression were assessed by reverse transcription-PCR in MCF-7 and MDA-MB-231 breast cancer cell lines., Results: Leptin and ObR were significantly overexpressed in primary and metastatic breast cancer relative to noncancer tissues. In primary tumors, leptin positively correlated with ObR, and both biomarkers were most abundant in G3 tumors. The expression of leptin mRNA was enhanced by insulin and hypoxia in MCF-7 and MDA-MB-231 cells, whereas IGF-I and estradiol stimulated leptin mRNA only in MCF-7 cells. ObR mRNA was induced by insulin, IGF-I, and estradiol in MCF-7 cells and by insulin and hypoxia in MDA-MB-231 cells., Conclusions: Leptin and ObR are overexpressed in breast cancer, possibly due to hypoxia and/or overexposure of cells to insulin, IGF-I, and/or estradiol.

Aims and Background: Vascular endothelial cadherin (VE-cadherin) preserves the tightness of the mature vascular network as a component of endothelial adherens junctions. Vascular endothelial growth factor (VEGF) makes VE-cadherin dissociate from complexes with beta-catenin, so that endothelial cells can loosely proliferate and migrate. We searched for relationships between VEGF and VE-cadherin levels in preoperative sera of patients with colorectal cancer (CRC). We also compared VE-cadherin levels of control and preoperative CRC sera in relation to clinicopathological features., Methods: We measured with an ELISA kit the serum levels of the proteins in preoperative samples from 125 CRC patients and in samples from 16 healthy volunteers., Results: Serum VE-cadherin was about fourfold higher in CRC patients than in controls (P < 0.00001), with similar results being found in subgroups with different clinicopathological features versus controls. VE-cadherin was not correlated with VEGF in the entire group of CRC patients nor in the subgroups of node-positive and node-negative patients, different grades of histological differentiation (G2 or G3), extent of tumor growth (pT1+pT2 or pT3+pT4), histopathological type (adenocarcinoma or mucinous carcinoma), sex, age, and tumor site (colon or rectum). However, the serum levels of VE-cadherin and VEGF in CRC patients, which were higher than the mean values of controls, tended towards a negative correlation in node-positive patients (P = 0.078, r = -0.279)., Conclusions: VEGF and VE-cadherin seem to be independent markers of angiogenesis in CRC with no significant correlation between their serum levels.

Purpose: Histological subtypes of uterine leiomyomas may substantially differ in their cellular biology, including the intensity of synthesis of cancer markers and expression of cell proliferation markers. The present investigation aimed to determine the activity of cancer procoagulant (CP) in subtypes of leiomyomas, including cellular leiomyomas, and to verify whether these activities correlate with immunoexpression of cell proliferation markers: the proliferating cell nuclear antigen (PCNA) and Ki-67., Materials and Methods: Preoperative peripheral venous blood and postoperative tissue material were obtained from 24 women operated on in a tertiary referral academic department. The activity of CP in serum was measured with the use of a coagulative method according to Gordon and Benson, and in tissue homogenates with the use of a spectrophotometric method according to Colucci et al. The control serum values were obtained from 20 healthy women without any gynecological disease, and the control solid tissue values from histologically confirmed postoperative normal reproductive tissues obtained from six patients. PCNA and Ki-67 expression were determined immunohistochemically using monoclonal antibodies., Results: Both the tissue and serum activity for CP was considerably higher for common leiomyomas and cellular leiomyomas than for control tissues, but did not differ significantly between the leiomyoma subtypes. Intratumor CP activity significantly correlated with PCNA expression but not with Ki-67 expression., Conclusions: Cellular leiomyomas do not differ substantially in the serum and intratumor CP activity from common leiomyomas. There is a relationship of intratumor CP activity with PCNA expression, a finding which requires further investigation.

EPO is known as an inducer of maturation and proliferation of erythrocytes. Moreover, it favours angiogenesis. In several studies it was encountered that EPO is a trophic agent that mediates survival and inhibits apoptosis of hypoxia affected cells, particularly those which build masses of irregularly vascularized cancers. The main task concerning EPO for oncologists is the choice to give or not to give recombinant EPO to anemia endangered cancer patients. EPO can do the quality of life better and cause recovery from anemia post chemotherapy and radiation of cancer patients. Nevertheless, EPO therapy shortens survival of patients in some cancers, in which antiapoptotic effect of EPO predominates directly in malignant cells. Thus, separately in every type of cancer, therapeutic use of recombinant EPO calls for prior investigations, if EPO signaling causes proliferation of cancer cells by direct stimulation of EPOR positive malignant cells. Unless the proliferative effect of EPO on cancer cells is excluded, its use in the therapy of anemia in cancer patients is not quite safe.

Leptin restricts intake of calories as a satiety hormone. It probably stimulates neoplastic proliferation in breast cancer, too. Growth of malignant cells could be regulated by various leptin-induced second messengers like STAT3 (signal transducers and activators of transcription 3), AP-1 (transcription activator protein 1), MAPK (mitogen-activated protein kinase) and ERKs (extracellular signal-regulated kinases). They seem to be involved in aromatase expression, generation of estrogens and activation of estrogen receptor alpha (ERalpha) in malignant breast epithelium. Leptin may maintain resistance to antiestrogen therapy. Namely, it increased activation of estrogen receptors, therefore, it was suspected to reduce or even overcome the inhibitory effect of tamoxifen on breast cell proliferation. Although several valuable reviews have been focused on the role of leptin in breast cancer, the status of knowledge in this field changes quickly and our insight should be continuously revised. In this summary, we provide refreshed interpretation of intensively reported scientific queries of the topic.

Diversity of P53 impact on tumor angiogenesis is due to the fact that wild-type P53 decreases expression of vascular endothelial growth factor (VEGF), but mutant P53 upregulates it. Therefore, we aimed at uncovering relations between preoperative serum levels of VEGF and P53 in colorectal cancer (CRC) patients. Preoperative blood samples of 125 CRC patients and 16 control healthy volunteers were examined with an ELISA-kit for serum P53 levels and VEGF. P53 did not correlate with VEGF in the whole group of CRC patients. However, P53 associated with VEGF in case of colorectal cancer patients, whose serum values of VEGF were higher than in controls (VEGF{H} >5.9333 pg/ml) (r=0.274, p<0.009). We revealed a positive correlation between P53 and VEGF{H} in subsets of poorly differentiated (G3) cancers (p<0.02), lymph node positive (p<0.007), pT3 or pT4 patients (p<0.004) without analogous relation in moderately differentiated (G2) tumors, node negative patients or pT1 or pT2 patients. P53 and IGF-I negatively correlated in all CRC patients (p<0.04) and VEGF{H} individuals of pT3 or pT4 (p<0.05) without any significant linkage in tumors of pT1 or pT2. The positive correlation between serum P53 and VEGF points at mutation of P53 and is a highly probable sign of poor prognosis in colorectal cancer. For now it can not be excluded that the binary analysis of serum P53 and VEGF could help select CRC patients endangered by rapid growth and lymph node metastases.

To elucidate the molecular profile of oestrogen receptors alpha and beta (ERalpha, ERbeta) we studied ERalpha and ERbeta expression at the mRNA and protein levels using real-time polymerase chain reaction (RT-PCR), Western blot analysis and immunohistochemical (IHC) methods in 41 primary breast cancers and surrounding tissues. ERalpha mRNA and ERbeta mRNA were detected in all of the breast cancer and normal matched tissues analysed. ERalpha mRNA levels showed greater diversity than ERbeta mRNA levels and the range of amount of ERbeta transcripts was far smaller than that of ERalpha. At the protein level, the percentage of ERalpha- or ERbeta-positive cases changed. Seventy percent of the tumours studied produced full-length 65 kDa ERalpha protein in Western blot analysis and 67% of assessed cases were positive in IHC. Full-length 57 kDa ERbeta protein was detected by Western blotting in 97% of analysed breast cancers, while 67% were ERbeta-positive using IHC. ERalpha was localised in the nucleus, while cytoplasmic and perinuclear localisation of ERbeta was observed in normal as well as in breast cancer cells. The amount of ERalpha (but not ERbeta) increased with age. The expression of ERalpha correlated positively with progesterone receptor and negatively with proliferation marker Ki-67. These results confirm the previous observations that the lack of ERalpha protein expression is not due to lack of ERalpha gene expression or methylation of ERalpha promoter, but due to post-transcriptional or post-translational mechanisms. Our investigation also suggests that ERalpha is more dysregulated in breast cancer, and thereby ERbeta is more tightly regulated in the tumour.

Human papilloma virus (HPV) infection and loss of P53 function have been identified as frequent events in various human tumors. The aim of this study was to evaluate P53 protein expression and to detect HPV in the tissue samples of 45 benign (papillomas) and 38 malignant conjunctival and eyelid lesions (27 basal cell carcinomas and 11 squamous cell carcinomas). We also looked for eventual relationships between P53 expression and clinicopathological features such as age, histological type of tumor, grading and staging. HPV infection was detected using the PCR-RFLP method. Specific primers were engaged and PCR products of HPV 16, 18, and 33, underwent enzymatic digestion at 37 degrees C. We revealed P53 protein expression in 30 out of 45 (66.6%) squamous cell papillomas. In the SCC and BCC groups, P53 was present in 31 out of 38 carcinomas and there was a statistically significant correlation between histological type of tumor and P53 protein expression. Malignant type HPV 16 and 18 were detected in three squamous cell papillomas, two BCCs and one SCC. However, we observed P53 protein expression in only two HPV-positive papillomas and one infiltrative type of BCC. P53 is probably involved in the development of conjunctival and eyelid tumors due to its high rate of presence in both benign and malignant neoplasms of these organs. HPV seems to occur rarely. In some cases its role in the pathogenesis of conjunctival and eyelid tumorigenesis should be considered as auxiliary.

Cancer cells perform their malicious activities through own cell membranes that screen and transmit inhibitory and stimulatory signals out of the cells and into them. This work is focused on changes of phospholipids content (PI-phosphatidylinositol, PS-phosphatidylserine, PE-phosphatidylethanolamine, PC-phosphatidylcholine) and electric charge that occur in cell membranes of colorectal cancer of pT 3 stage, various grades (G2, G3) and without/with metastasis. Qualitative and quantitative composition of phospholipids in the membrane was determined by HPLC (high-performance liquid chromatography). The surface charge density of colorectal cancer cell membranes was measured using electrophoresis. The measurements were carried out at various pH of solution. It was shown that the process of cancer transformation was accompanied by an increase in total amount of phospholipids as well as an increase in total positive charge at low pH and total negative charge at high pH. A malignant neoplasm cells with metastases are characterized by a higher PC/PE ratio than malignant neoplasm cells without metastases., ((Mol Cell Biochem 276: 113-119, 2005).)

Apoptosis is a process associated with development and progression of breast cancer. The association between connexins and programmed cell death has only been demonstrated in a few studies. The aim of the present study was the evaluation of Cx26 and Cx43 expression in breast cancer in correlation with Bcl-2 and Bak proteins as well as with selected clinicopathological features. Tissue samples from 71 women were examined by immunohistochemistry, using the streptavidin-biotin-peroxidase complex technique for Cx26, Cx43, Bak and Bcl-2. Cytoplasmic expression of Cx26 and Cx43 was detected in 34 (47.9%) and 61 (85.9%) of breast cancers, respectively. Bcl-2 and Bak expression was found in 59 (83%) and 50 (70%) of studied cases, respectively. We found a positive correlation between Cx26 and Bak expression (r=0.541, p<0.0001) as well as between Cx43 and Bak (r=0.589, p<0.0001), but not between the studied connexins and Bcl-2. The expression of Cx26 was not associated with age, tumor size, lymph node status or histological grade. However, we observed an association between Cx43 expression and histological grade G3 (p<0.04). Cytoplasmic localization of evaluated connexins supports the concept of alterations in connexins expression in breast cancer cells. The associations of evaluated connexins with Bak protein could suggest that connexins localized in the cytoplasm may participate in signaling apoptotic pathways.

Aim: Proteolytic degradation of the extracellular matrix facilitates cancer invasion and promotes metastasis. The study aims at evaluation of preoperative and postoperative serum cathepsins B and D levels in correlation with selected anatomoclinical features of colorectal cancer., Methods: Blood samples were collected from 63 colorectal cancer patients before curative operation of the tumor 10 d later. Blood that was obtained from 20 healthy volunteers, served as a control. The activity of cathepsin B was measured with Bz-DL-arginine-pNA as a substrate at pH 6.0, while cathepsin D activity was determined with urea-denatured hemoglobin (pH 4.0)., Results: The preoperative and postoperative activities of cathepsin B were significantly (P<0.00001) lower in serum of colorectal cancer patients than in control group. However, postoperative values of this protease were significantly increased in comparison with preoperative ones (P = 0.031). Activity of cathepsin D appeared to be significantly higher in colorectal cancer sera (P<0.00001) compared with controls. No statistically significant differences between preoperative and postoperative activity of cathepsin D were noted (P = 0.09). We revealed a strong linkage of cathepsins' levels with lymph node status and pT stage of colorectal cancer., Conclusion: Blood serum activities of cathepsin B and D depend on the time of sampling, tumor size and lymph node involvement. Significantly, increased activity of cathepsin D could indicate a malignant condition of the large intestine. In our work, the serum postoperative decrease of cathepsin B activity appears as an obvious concomitant of local lymph node metastasis-the well-known clinicopathological feature of poor prognosis.

Disturbance in expression of estrogen receptors together with changing influence of growth factor receptors and apoptosis associated proteins plays a role in breast cancer development and progression. However, immunohistochemical detection and relationships among these proteins were not often considered in relation to breast cancer and a few evaluations of expression provided mismatching results and conclusions. Consequently, we examined by immunohistochemistry the expression of the insulin-like growth factor-I receptor (IGF-IR), estrogen receptor alpha (ERalpha) and apoptosis-associated proteins, Bcl-2 and Bax, in human primary breast cancer, as well as analyzing the relationships among these proteins. The positive immunostaining for IGF-IR, ERalpha, Bcl-2 and Bax was noted in 56, 63.8, 82.8 and 50% of tumors, respectively. We observed that IGF-IR negatively correlated with ERalpha in the group of all tumors and in axillary node negative cancer (p<0.03, p<0.05, respectively), but not in the subgroup of node positive cancer. Expression of ERalpha correlated positively with Bcl-2 and negatively with Bax proteins (p<0.0001, p<0.05, respectively). We did not note significant relationships between IGF-IR and Bcl-2, or IGF-IR and Bax proteins. We found that increased Bax expression was associated with positive lymph node status, pT2 stage and G3 grade of tumors. Knowledge about alterations in the IGF-IR expression and relations of the receptor to other biological factors could help in our understanding of breast cancer biology and the importance of the IGF-IR in cancer progression as well as in effective management of breast cancer.

Aim: To evaluate of Cx26 in correlation with Bcl-xL and Bax proteins in colorectal cancer., Methods: Immunohistochemical staining using specific antibodies was performed to evaluate the protein expression of Cx26, Bax and Bcl-xL in 152 colorectal cancer samples and the correlations among studied proteins as well as the relationships between the expression of Cx26, Bax, Bcl-xL and clinicopathological features were analyzed., Results: Both normal epithelial cells and carcinoma cells expressed Cx26, Bax and Bcl-xL, but Cx26 in cancer cells showed aberrant, mainly cytoplasmic staining. Expression of Cx26, Bax and Bcl-xL was observed in 55.9%, 55.5% and 72.4% of evaluated colorectal cancers respectively. We found the positive correlation between Cx26 and Bax expression (r = 0.561, P<0.0001), Cx26 and Bcl-xL (r = 0.409, P<0.0001) as well as between Bax and Bcl-xL (r = 0.486, P<0.0001). Association of Cx26, Bax and Bcl-xL expression with histological G2 grade of tumors was noted (P<0.005, P<0.001 and P<0.002 respectively)., Conclusion: Cytoplasmic presence of Cx26 and its association with apoptotic markers could indicate a distinct role from physiological functions of Cx26 in cancer cells and it could suggest that connexins might be a target point for modulations of apoptosis with therapeutic implications.

Cancer development is essentially a tissue remodeling process in which normal tissue is substituted with cancer tissue. A crucial role in this process is attributed to proteolytic degradation of the extracellular matrix (ECM). Degradation of ECM is initiated by proteases, secreted by different cell types, participating in tumor cell invasion and increased expression or activity of every known class of proteases (metallo-, serine-, aspartyl-, and cysteine) has been linked to malignancy and invasion of tumor cells. Proteolytic enzymes can act directly by degrading ECM or indirectly by activating other proteases, which then degrade the ECM. They act in a determined order, resulting from the order of their activation. When proteases exert their action on other proteases, the end result is a cascade leading to proteolysis. Presumable order of events in this complicated cascade is that aspartyl protease (cathepsin D) activates cysteine proteases (e.g. cathepsin B) that can activate pro-uPA. Then active uPA can convert plasminogen into plasmin. Cathepsin B as well as plasmin are capable of degrading several components of tumor stroma and may activate zymogens of matrix metalloproteinases, the main family of ECM degrading proteases. The activities of these proteases are regulated by a complex array of activators, inhibitors and cellular receptors. In physiological conditions the balance exists between proteases and their inhibitors. Proteolytic-antiproteolytic balance may be of major significance in the cancer development. One of the reasons for such a situation is enhanced generation of free radicals observed in many pathological states. Free radicals react with main cellular components like proteins and lipids and in this way modify proteolytic-antiproteolytic balance and enable penetration damaging cellular membrane. All these lead to enhancement of proteolysis and destruction of ECM proteins and in consequence to invasion and metastasis.

Colorectal cancer is the third most often cause of morbidity and mortality due to cancer in Poland. Thromboembolic complications are common events during the course of the disease. It is well known that hemostatic proteins play an important role in cancer progression. The purpose of the study was to evaluate the in loco interactions among colorecatal cancer and coagulation factors. 21 cases of G2 colorectal adenocarcinoma obtained during surgical resection were examined. Immunohistochemical procedures according to ABC method were employed. Tissue factor (TF) and coagulation factors II, VII, X, IX were observed in cancer cells and except factors II and IX--in tumor associated macrophages. TF was also demonstrated in endothelial cells of small blood vessels. Strong expression of fibrinogen was observed among connective tissue at some distance around malignant tumor while weaker expression was found in tumor stroma. Expression of F(1+2), the by-product of thrombin generation, was revealed in cancer cells, macrophages and in the tumor stroma. The results indicate extravascular activation of blood coagulation in loco in colorectal cancer that is TF-dependent.

Aim: Reactive oxygen species (ROS) can induce carcinogenesis via DNA injury. Both enzymatic and non-enzymatic parameters participate in cell protection against harmful influence of oxidative stress. The aim of the present study was to assess the levels of final lipid peroxidation products like malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) in primary colorectal cancer. Moreover, we analysed the activity of main antioxidative enzymes, superoxide dismutase (Cu, Zn-SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GSSRG-R) and the level of non-enzymatic antioxidants (glutathione, vitamins C and E)., Methods: Investigations were conducted in 81 primary colorectal cancers. As a control, the same amount of sample was collected from macroscopically unchanged colon regions of the most distant location to the cancer. Homogenisation of specimens provided 10% homogenates for our evaluations. Activity of antioxidant enzymes and level of glutathione were determined by spectrophotometry. HPLC revealed levels of vitamins C and E and served as a method to detect terminal products of lipid peroxidation in colorectal cancer., Results: Our studies demonstrated a statistically significant increase in the level of lipid peroxidation products (MDA-Adc.muc.-2.65+/-0.48 nmol/g, Adc.G3-2.15+/-0.44 nmol/g, clinical IV stage 4.04+/-0.47 nmol/g, P<0.001 and 4-HNE-Adc.muc. -0.44+/-0.07 nmol/g, Adc.G3-0.44+/-0.10 nmol/g, clinical IV stage 0.52+/-0.11 nmol/g, P<0.001) as well as increase of Cu,Zn-SOD (Adc.muc.-363+/-72 U/g, Adc.G3-318+/-48 U/g, clinical IV stage 421+/-58 U/g, P<0.001), GSH-Px (Adc.muc. -2143+/-623 U/g, Adc.G3-2005+/-591 U/g, clinical IV stage 2467+/-368 U/g, P<0.001) and GSSG-R (Adc.muc.-880+/-194 U/g, Adc.G3-795+/-228 U/g, clinical IV stage 951+/-243 U/g, P<0.001) in primary tumour comparison with normal colon (MDA-1.39+/-0.15 nmol/g, HNE-0.29+/-0.03 nmol/g, Cu, Zn-SOD-117+/-25 U/g, GSH-Px-1723+/-189 U/g, GSSG-R-625+/-112 U/g) especially in mucinous and G3-grade adenocarcinomas as well as clinical IV stage of colorectal cancer. We also observed a decrease of CAT activity (Adc.muc. -40+/-14 U/g, clinical IV stage 33+/-18 U/g vs 84+/-17 U/g, P<0.001) as well as a decreased level of reduced glutathione (clinical IV stage 150+/-48 nmol/g vs 167+/-15 nmol/g, P<0.05) and vitamins C and E (vit. C-clinical IV stage 325+/-92 nmol/g vs 513+/-64 nmol/g, P<0.001; vit. E-clinical IV stage 13.3+/-10.3 nmol/g vs 37.5+/-5.2 nmol/g)., Conclusion: Colorectal carcinogenesis is associated with serious oxidative stress and confirms that gradual advancement of oxidative-antioxidative disorders is followed by progression of colorectal cancer.

In our previous investigation Insulin Receptor Substrate 1 (IRS-1) correlated with proliferation marker Ki-67 in human breast cancer. The aim of the present study was to assess relationships between IRS-1 expression and anti-apoptotic Bcl-xL as well as proapoptotic Bax proteins, assessed by immunohistochemistry, in primary tumors and lymph node metastases of breast cancer. IRS-1 is positively associated with both Bcl-xL and Bax in primary and metastatic tumors. Thus, our results could suggest that IRS-1 might affect turnover of cancer cells and breast cancer progression through activation of mitogenesis and participation in the regulation of the balance between anti- and proapoptotic pathways.

Background: Gap junctional intercellular communication (GJIC) is a mechanism for direct cell-to-cell signalling and is mediated by gap junctions, which consist of transmembrane proteins called connexins (Cxs). Human colorectal epithelial cells express Cx32 and Cx43., Methods: Tissue samples (152 from colorectal cancer and 75 from adenoma) were investigated by immunohistochemistry, using the antibody for Cx26. Moreover, Cx26 was assessed in normal epithelium of the colon and rectum, adjacent to colorectal cancer., Results: In normal epithelium and adenomas, intercellular, punctate staining for Cx26 was observed. In adenomas with severe dysplasia, focally decreased expression of Cx26 was observed. Among 152 colorectal cancers, 55.9% classified only as adenocarcinoma stained positive for Cx26, but mainly cytoplasmic staining was found. We observed a positive correlation between Cx26 expression and tumor G2 grade (p < 0.005). The expression of Cx26 did not correlate with age, sex of patients, tumor localization, lymph node status or tumor size., Conclusions: Our results show that during colorectal carcinogenesis, loss of normal intercellular Cx expression occurs. Furthermore, the cytoplasmic presence of Cx26 could indicate a different role of Cx26 in neoplastic cells than participation in GJIC., (Copyright (c) 2005 S. Karger AG, Basel.)

Purpose: It is currently believed that cancer procoagulant (CP), an enzymatic protein, is a product of malignant neoplastic cells. The present study was designed to test whether it is also synthesized by benign neoplastic cells, namely uterine leiomyomas., Materials and Methods: We determined the activity of CP in the blood serum of women with uterine leiomyomas (N = 24), normal women (N = 15), and genital cancer patients (N = 6) by the coagulative method according to Gordon and Benson. Also, the CP activity in 10% tissue homogenates of uterine leiomyomas, normal uterine muscle and tissues of cervical and endometrial carcinoma was determined by the chromogenic method according to Colucci et al., Results: The mean CP activity in the sera of women with uterine leiomyomas was 181.1 seconds (s) +/- 19.9 s, in healthy women--293.2 s +/- 33.8 s, and in genital cancer patients--78.8 +/- 18.5 s (all differences: p < 0.001). Similarly, in homogenates of uterine leiomyomas the CP activity was 19.6 +/- 3.8 nmoles pNa/ml, in normal uterine muscle it was 13.2 +/- 2.2 nmoles pNa/ml, and in cancerous tissue--28.0 +/- 6.6 nmol pNa/ml (all values being significantly different from each other). There was a strong correlation (r = -0.8122; p < 0.001) between the CP activity in uterine leiomyomas and serum activity, suggesting that the source of the serum CP activity was from the leiomyoma. The coagulation time of 120 to 240 s by the Gordon and Benson method supported the diagnosis of uterine leiomyoma, and a value below 120 s--the suspicion of genital cancer., Conclusions: Uterine leiomyomas, representing benign genital neoplasia, synthesize CP and are the likely origin of CP activity in blood, as has been described for malignant tumors, but to a lesser degree. There may be a role for CP as a tumor marker of genital neoplasia.

Mast cells may play a role in pathogenesis of the otitis media with effusion (OME) in children. The study involved 72 children with no history of allergic diseases. The analysed material were adenoids removed on the grounds of hypertrophy. Immunohistochemical analyses were carried out using antibodies (Dako, M 7052) directed against mast cell tryptase. The slides evaluation was performed by means of optic microscope. The presence of mast cells within adenoidal tissue were scored. There were no statistically significant difference between the numbers of mast cells in each examined adenoid as well as age groups. Our results correspond with those obtained by other authors according to mast cells distribution in adenoid tissue and do not indicate any particular role of mast cells in pathogenesis of OME in children.

Cell proliferation and programmed cell death are considered to be important events in carcinogenesis. The object of our study was to evaluate the expression of the Bcl-2 protein family (Bcl-2, Bak, Bax), p53, proliferating cell nuclear antigen (PCNA), and Ki-67 protein immunoexpression as well as the correlation between the examined markers and some clinicopathological features in papillomas and cancers of conjunctiva and eyelid. Forty-five squamous cell papillomas (SCP), 11 squamous cell cancers (SCC), and 27 basal cell cancers (BCC) were estimated. In the SCP group, p53 protein expression was observed in 30 cases (66.6%), Ki-67 in 14 (31.1%), PCNA in 44 (97.8%), Bcl-2 in 24 (53.3%), Bak in 28 (62.2%), Bax in 31 (68.9%), and Bcl-xl in 11 (100%). In the SCC group, p53 protein expression was evaluated in 8 cases (72.8%), Ki-67 in 2 (18.2%), PCNA in 8 (72.7%), Bcl-2 in 5 (45.4%), Bax and Bak both in 10 (90.9%), and Bcl-xl in 100%. In the BCC group, p53 protein expression was estimated in 23 cases (85.1%), Ki-67 in 13 (48.1%), PCNA in 26 (96.2%), Bcl-2 in 13 (48.1%), Bak in 21 (77.8%), Bax in 22 (81.5%), and Bcl-xl in 23 (85.2%). We observed a correlation between some clinicopathological features and the examined markers of apoptosis and cell proliferation, which seemed to be important events in cancer development.

Insulin-like growth factor-I (IGF-I) and IGF-I receptor (IGF-IR), despite their well-known roles in cell survival and proliferation, can also weakly enhance apoptosis. To study the relationships between the IGF-IR and Bax as well as Bak, 144 cases of colorectal cancer were examined by immunohistochemistry, using the avidin-biotin-peroxidase method. The results were correlated with selected clinicopathological features of colorectal cancer and with the expression of IGF-IR, Bax, and Bak in normal colon mucosa. In Bax-, Bak-, or IGF-IR-positive cancers, the adjacent colorectal mucosa revealed positive immunostaining for these proteins. In the majority of Bax-, Bak-, or IGF-IR-negative tumors, we observed no staining for these proteins in adjacent mucosa. The strong immunostaining for IGF-IR, Bax, and Bak was noted in 50.8, 55.5, and 49.3% of tumors, respectively. We observed positive correlations between IGF-IR and Bax (P < 0.002, r = 0.302), between IGF-IR and Bak (P < 0.0001, r = 0.407), and between Bax and Bak (P < 0.0001, r = 0.474). No relationship was noted between IGF-IR expression and tumor grade, stage, or lymph node status. We found negative associations between Bax, Bak, and tumor grade (P < 0.01 and P < 0.003, respectively), but no relationships between Bax and Bak and tumor stage or between Bax and Bak and lymph node status. Our results demonstrate that, in addition to overexpressed IGF-IR, there are relationships between IGF-IR and proapoptotic proteins in colorectal carcinomas that could contribute to increased cell turnover and the progression of colorectal cancer.

We used immunohistochemistry to compare the expression of estrogen receptors (ERalpha and ERbeta) in term myometria of 32 pregnant women divided in two groups. Group I comprised of 16 women in labour and group II included 16 non-laboring gravidas. We observed cytoplasmatic localization of both ER isoforms and no differences in the ER expression between the two groups of patients. The abundance and specific localization of ERs in human term myometrium seems to be independent of its contractile activity which may point to the specific role of those receptors in late pregnancy myometrium.

Objectives: Cancer procoagulant (CP) is a sulfhydryl proteinase thought to be synthesized mainly by neoplastic cells. Consequently, increased CP activity in blood serum was interpreted as being associated with the presence of a proliferative process in the host's body. To date, CP activity has not been systematically studied in cases of genital carcinoma. The present study is aimed at evaluation of CP activity in women with genital carcinoma., Design: A case-controlled study backed up by histopathological examination., Materials and Methods: Peripheral blood was sampled preoperatively in a sterile manner from an antecubital vein, from 16 women with cervical carcinoma and 15 women with uterine carcinoma. Blood for the reference group of 12 healthy women was obtained in an identical manner after an overnight fast. The CP activity in serum was determined using the coagulative method according to Gordon and Benson, and was expressed as coagulation time in seconds (s). The CP activity in 10% tissue homogenates (in saline) of genital cancer was determined by the chromogenic method according to Colucci et al., Results: The mean CP activity in serum of women with cervical carcinoma (78.28 +/- 15.25 s) and of women with uterine carcinoma (79.63 +/- 12.02 s) was significantly different (P < 0.0001) from the respective values found in healthy women (281.33 +/- 43.19 s). The CP activity in neoplastic tissue was 28.50 +/- 6.40 nmol pNa/mL for cervical carcinoma, and 28.31 +/- 3.92 nmol pNa/mL for uterine carcinoma, both values being significantly higher (P < 0.0009) than the activity found in the normal tissues. There was no established relationship between neoplastic CP activity and FIGO staging of the disease., Conclusions: This is the first study to demonstrate the concomitant presence of CP activity in serum and neoplastic tissue of women with genital carcinoma. These patients have decreased coagulation time and thus are likely to develop coagulation disturbances in the course of their cancer. There may be a role for CP as a tumor marker of genital carcinoma.

Tamoxifen and raloxifene are widely used in clinical practice. It has been found that tamoxifen treatment increases the risk of development of endometrial cancer. The effects of tamoxifen and raloxifene on endometrium might be caused by different estrogen receptor expression. The aim of the present study was immunohistochemical evaluation of the effects of tamoxifen and raloxifene on estrogen receptors, and Ki-67 antigen expression in the human endometrial adenocarcinoma Ishikawa cell line. Tamoxifen in concentrations of 10 microM and 20 microM increased ERalpha expression without any effect on ERbeta. All used concentrations of tamoxifen and raloxifene (0.1 nM, 1 nM, 10 nM, 1 micro M, 10 microM and 20 microM) had no effect on expression of ERbeta. Tamoxifen, but not raloxifene, increased Ki-67 antigen expression in the Ishikawa cell line. Tamoxifen, in contrast to raloxifene, increased proliferation of endometrial adenocarcinoma cells as well as exerted the shift of ERalpha/ERbeta ratio. Thus, it could be responsible for increased carcinogenic effect during tamoxifen treatment.

Angiogenesis or the formation of new vessels out of pre-existing capillaries is a sequence of events that is fundamental to physiology of the female reproductive tract and also pathologic processes such as ovarian hyperstimulation syndrome. Many factors include vascular endothelial growth factor (VEGF), angiopoietins and others involved in the regulation of angiogenesis have been identified. There are some endocrine control mechanisms, which stimulate or inhibit the angiogenesis. The studies indicate that the normal processes of folliculogenesis, ovulation and corpus luteum function in the ovary and the control of menstruation and implantation in the endometrium are profoundly dependent on the angiogenesis. The rapid, controlled and cyclical nature of angiogenesis in the female reproductive tract suggests that interference with this process should provide a novel approach to manipulation of reproductive function.

Apoptosis and proliferation are processes associated with the development and progression of breast cancer. The sensitivity of tumour cells to the induction of apoptosis depends on the balance between pro- and anti-apoptotic proteins. The expression of Bak and Bcl-2 was examined using an immunohistochemical method in 71 primary breast cancers. Furthermore, Bcl-2 and Bak were assessed in the normal mammary gland as well as in benign mammary dysplasia adjacent to breast cancer. Positive immunostaining for Bcl-2 was observed in 77.8% of cases of normal breast epithelium (NBE), 93% of benign dysplasia without intraductal proliferation (BBD) as well as in 94% of intraductal proliferative lesions of the breast (BIPL). Expression of Bak was detected in 39% of cases of NBE, 45% of BBD and in 67% of BIPL. In breast cancer Bcl-2 and Bak expression was found in 83% and 70% of the cases studied, respectively. Increased Bcl-2 expression in primary tumours significantly correlated with favourable prognostic factors, namely pT1, G2 and lack of metastases to the regional lymph nodes (p < 0.01, p < 0.03, p < 0.02, respectively). There were no relationships between Bak and the clinicopathological features studied, but our results indicate changes in the expression of Bak during breast cancer development and progression. It would appear to be important to assess and compare pro- and anti-apoptotic proteins between normal mammary gland, benign mammary dysplasia and the primary tumours of breast cancer. This knowledge should be helpful in understanding breast cancer development and progression.

The experiment was performed on 36 Wistar rats. On the first day of the experiment iodoacetate was administered to the left posterior knee joint of the 18 rats which composed Group I. The second group of 18 rats received additionally doxycycline (doxy) through the gastric tube in doses comparable with those of doxycycline used in humans. The experiment lasted 21 days. The animals were sacrificed after 7, 14 and 21 days in groups of 6 rats each. In sections stained with Safranin 0 semiquantitative histochemical intensity tests were performed on articular cartilage glycosaminoglycans (GAG) using a four-point scale (0-3). In the first group examined destructive lesions in the articular cartilage and weak reactivity on GAG were noted at all stages of the experiment. The intensity of GAG staining was higher in the second group after 14 and especially after 21 days, which may suggest a protective action of doxy on articular cartilage., (Copyright 2004 Via Medica)

The study sought a correlation between the number of AgNOR granules and the degree of cervical intraepithelial neoplasia (CIN). Thirty-five sections (5 normal, 10 CIN1, 10 CIN2 and 10 CIN3) were subjected to retrospective analysis. The percentage of cells with 1, 2, 3, 4 and more AgNORs was calculated and the number of granules per 100 cells was counted. The number of cells containing single granules decreases. However, the number increases with CIN level when the cells contain 4 and more AgNORs. The number of granules per 100 cells also increases with the degree of CIN. It can be thus concluded that the number of cells with 4 and more AgNOR granules can serve as a CIN differentiation exponent., (Copyright 2004 Via Medica)

The success of current hormonal therapies used in breast cancer patients often depends on estrogen receptor (ER) status. The simultaneous pathological assessment of primary breast tumors (PTs) and metastases to regional lymph nodes (MRLNs) could improve the effectiveness of this treatment, but is rarely performed. Consequently, we studied by immunohistochemistry the expression of ERalpha (ERalpha), ERbeta (ERbeta), and the proliferation marker (Ki-67) in PTs and matching MRLNs. The expression of each ERalpha, ERbeta, and Ki-67 positively correlated between PTs and MRLNs (r=0.751, p<0.0001; r=0.391, p<0.03; and r=0.707, p<0.0001, respectively). A negative correlation between the expression of ERalpha and Ki-67 was found in PTs (r=-0.678, p<0.001) and MRLNs (r=-0.501, p<0.005). A negative correlation between ERalpha and Ki-67 was also observed in PTs vs. MRLNs (r=-0.619, p<0.0001). Moreover, the expression of ERalpha and Ki-67 was not associated with nodal status, contrary to ERbeta expression, which correlated with negative axillary node status. The results indicated that the associations among ERalpha, ERbeta, and Ki-67 are maintained in PTs and MRLNs. Thus, simultaneous assessment of selected markers in PT and MRLN might help in understanding the mechanisms of breast cancer progression as well as result in the development of new principles for diagnosis and individual therapy planning of estrogen dependent cancer.

The aim of our study was an evaluation of the expression of cell proliferation markers (PCNA and Ki-67) in conjunctival and eyelid papillomas and squamous and basal cell cancers. A series of 9 cases of squamous cell cancer (SCC), 15 cases of basal cell cancer (BCC) and 43 cases of squamous cell papilloma (SCP) were assessed using the immunohistochemical method with monoclonal antibodies. PCNA overexpression was observed in 100% of SCP, in 88.8% of SCC and in 100% of BCC cases. Ki-67 overexpression was seen in 32.5% of cases of SCP, in 22.2% of SCC and in 66.6% of BCC. The results showed that an evaluation of Ki-67 expression is the most valuable cell proliferation marker.

The aim of our study was to evaluate the immunohistochemical expression of Bcl-2 and Bax protein in optic nerve axons after severe eyeball injury and in the eyes with absolute glaucoma. A series of 19 eyeballs, enucleated because of absolute glaucoma and 41 eyeballs, enucleated, following extensive injury, at the Department of Ophthalmology of the Medical University of Bialystok, were taken into our study. The immunohistochemical reaction was performed with Bcl-2 and Bax protein antibodies and by the LSAB technique. DAB was used in order to visualise the reaction. The optic nerve axons in glaucomatous eyeballs showed statistically significant higher Bax protein expressions than those of Bcl-2 proteins. In the optic nerve axons after severe eyeball injury, a non-significantly higher Bcl-2 protein expression was observed.

Gap junctional intercellular communication (GJIC) is a mechanism for direct cell-to-cell signalling and is mediated by gap junctions (GJs), which consist of proteins called connexins (Cxs). GJIC plays a critical role in tissue development and differentiation and is important in maintenance of tissue homeostasis. The purpose of the study was to evaluate the expression of Cx26, Cx32 and Cx43 in the human colon. Surgical specimens were obtained from patients who underwent surgical resection of colorectal tumours. Tissue samples (50 cases) were collected from normal colon, at the maximum distance from the tumor. Using antibodies for Cx26, Cx32 and Cx43, immunohistochemical detection was made. In epithelial cells, strong Cx26 immunoreactivity was found, whereas Cx32 and Cx43 were sparsely distributed. Strong Cx43 immunostaining in muscularis mucosae was observed. In the circular layer of muscularis externa, expression of Cx43 and Cx26 was seen, but only in the portion closest to the submucosa. No immunoreactivity was found in the longitudinal muscle layer. Small vessels stained positively only for Cx43. Furthermore, there was no difference in staining between samples derived from various sections of the colon. This study showed immunohistochemically for the first time the expression of Cx26 in human colon mucosa.

We have undertaken an attempt to compare the application efficacy of the proliferative activity markers in differential diagnosis of thyroid Hürthle cell tumors (HCT) using the PCNA and Ki-67 labeling and AgNOR visualisation techniques. The present work is a retrospective analysis of 78 Hürthle cell tumors: 20 Hürthle cell carcinomas (HCC), 32 Hürthle cell adenomas (HCA) and 26 hyperplastic nodules with Hurthle cell metaplasia (HCM). Five microm sections were stained according to AgNOR technique and labeled with antibodies against PCNA and Ki-67. AgNOR dot count in the nucleus and proliferative index (PI - percentage of cells expressing PCNA and Ki-67) in randomly chosen nuclei (100 in case of AgNOR and over 1000 in case of PI) were evaluated in each slide. The mean values of AgNOR dot count, PI-PCNA and PI-Ki-67 in HCC, HCA and HCM were respectively: 5.1, 61.3 and 54.9; 3.4, 42.4 and 38.6 and 2.5, 39.3 and 34.3. Statistically significant difference was found in all the proliferative activity markers between malignant and benign tumors: HCC:HCA (p<0.01) and HCC:HCM (p<0.001). There was no statistically significant difference between HCA and HCM.

The aim of our study was to estimate morphometric parameters of synaptophysin (Syn-38) immunoreactive ganglion cells in colorectal cancer (within and at various distances from neoplastic infiltration) in postoperative material from 60 patients. We analysed the intensity of Syn-38 expression in Auerbach ganglion neurones, mean number of these cells in the ganglion, and their longitudinal and transverse diameters. The results showed a statistically significant reduction in the number of neurones in intramural ganglia of the large intestine located in neoplastic infiltration and in its close proximity. The size of ganglion cells was directly proportional to the distance from cancer infiltration and inversely proportional to Syn-38 content, which may be explained by degenerative changes and dysfunction of these cells. This correlation was significant in the case of cells with the cytoplasmatic Syn-38 immunoreactivity pattern, but did not refer to the cells with perimembranous pattern, which seemed to be undamaged. Morphometric analysis of synaptophysin immunoreactive ganglion cells in Auerbach plexus in colorectal cancer may be a new useful marker for the description of changes in the intestinal nervous system as well as a prognostic factor for colorectal cancer.

In 36 Wistar rats with the iodoacetate-induced experimental osteoarthrosis (OA), effects of doxycycline, given orally, were determined on histochemical reactions of glycosaminoglycans (GAG) in the epiphyseal plate cartilage. The epiphyseal plate of rats with OA was reduced in height (especially the proliferative zone), cell columns were disorganized, many chondrocytes were irregular and polygonal, their nuclei were pycnotic, the intensity of GAG staining was irregular and predominantly reduced, which can be interpreted as signs of degeneration. A concomitant administration of doxycycline in the second group of rats prevented, to some extent, the negative effects of iodoacetate on chondrocytes and led to a more pronounced intensity of GAG reactions in the matrix of the epiphyseal plate.

Purpose: The aim of our study was the evaluation of Bcl-2, Bcl-xl, Bak and Bax immunoexpression in axons of the optic nerve with absolute glaucoma., Material and Methods: This study was conducted on 19 patients with absolute glaucoma, suffered from severe ophthalmalgia, treated of necessity by enucleation in 1991-2003 in our Department of Ophthalmology. The samples were immuno-stained with antibodies for Bcl-2, Bak, Bax and Bcl-xl protein. The reactions were performed in Labelled Streptavidin Avidin Biotin (LSAB) technique. The evaluation of immuno-staining was calculated as the percentage of cells with positive reaction to the total number of cells encountered in 10 representative fields, by light microscopy using a x 20 objective lens. Scores were based on the following scale: (++), over 50% of the field showing positive immuno-staining, (+) 10-50% with positive immunostaining., Results: In our study proapoptotic Bak and Bax protein expression was stronger than antiapoptotic Bcl-2, Bcl-xl protein expression; Bak/Bcl-2 (p = 0.008), Bax/Bcl-2 (p = 0.0007), Bak/Bcl-xl (p = 0.0356), Bax/Bcl-xl (p = 0.0077)., Conclusions: The results of our study demonstrate high proapoptotic trends in axons of the optic nerve in the stage of absolute glaucoma.

We attempted to describe a GLUT-1 expression in breast cancer and characterize correlation between GLUT-1 and ERs alpha and beta expression as well as correlate this with clinicopathologic features. Sixty-nine patients were involved in the study. GLUT-1, ER-alpha and ER-beta immunocytochemistry was performed using the streptavidin- biotin method. Thirty-seven (53.6%) out of total 69 were GLUT-1 positive. Of GLUT- 1 positive 45.3% were ER-alpha-positive, whereas 81.3% of ER-alpha-negative were GLUT-1 positive. Statistically significant correlation was observed between GLUT-1 and ER-alpha expression status but neither between GLUT-1 and ER-beta nor with clinicopathologic features. No statistically significant correlation was found between expression level (expressed as immunocytoreactive score) of GLUT-1, ER-alpha and ER-beta. Since most of ER-alpha-negative (81.3%) were GLUT-1 positive and significant correlation exists between the two receptors it is reasonable to assume that some functional relation might exists between the expression of two receptors.

The aim of our study was an evaluation of Bcl-xl and Bak protein expressions in optic nerve axons in eyeballs after severe injury and absolute glaucoma. We examined a series of 41 eyeballs, which were enucleated, following extensive injury and a series of 19 eyeballs from patients with absolute glaucoma. The immunohistochemical reaction was performed, using antibodies against human Bcl-xl and Bak protein with LSAB and DAB. In the injured eyeballs in Group I, Bcl-xl protein expression was observed in 53.8%, Bak in 38.5%, in Group II, Bcl-xl in 40%, Bak 55%; in Group III, Bcl-xl in 62.5%, Bak in 62.5%. Nine (9), out of 19 (47.4%) cases showed Bcl-xl protein positivity, Bak 15, out of 19 (78.9%). The percentage of cases with Bak protein positivity was statistically higher than that for Bcl-xl (Bak/Bcl-xl p=0.0356). The results showed that there may be a dominance expression of proapoptotic proteins in optic nerve axons in glaucoma.

The aim of this study was to check if the expression of CD44v4 in epithelial cells of the colorectal cancer correlates with the pTN stage and the histopathological grade of malignancy--G. Samples of tumour tissue (TT), as well as those of healthy tissue (HT) and of tumour adjacent tissue (TAT) were obtained from 25 patients. An evaluation of the expression of CD44v4 was performed in a flow cytometer. The mean value of the percentage of epithelial cells with co-expression of CD44v4 was lower in pT2 stage than that in pT3 only in HT. The expression of CD44v4 in epithelial cells was higher in cases without lymph node metastases only in TAT. The expression of CD44v4 in epithelial cells was higher in G2 than in G3 degree only in TAT as well. According to the obtained results, it is difficult to state if CD44v4 can influence the progress of colorectal cancer.

The balance between cell proliferation and programmed cell death plays a crucial role in malignant development. Bcl-2 family proteins, including proapoptosis protein Bak and antiapoptosis protein Bcl-2, regulate the apoptotic process. Mutation of the p53 gene, which results in P53 protein accumulation, was observed in many types of human cancer. The aim of our study was to evaluate immunohistochemical Bcl-2, Bak, and P53 protein expression and the relation between these proteins in conjunctival and eyelid benign and malignant tumors. We examined a series of 42 papillomas (CEP), 12 squamous cell cancers (SCC), and 19 cases of basal cell cancer (BCC). The age in the CEP group ranged from 18-94 years, and in the SCC and BCC groups from 42-87 years. Staining patterns were correlated with sex, age, and tumor localization. P53 protein-positive immunostaining was observed in 71% of cases, Bcl-2 in 83.9%, and Bak in 74.2 cases in the SCC and BCC groups. In the CEP group, P53 overexpression was observed in 90.5% of cases, Bcl-2 in 71.4%, and Bak in 76.2%. No statistically significant correlation was found between examined protein expression and sex, age, and tumor localization. An inverse correlation was observed between P53 and Bak protein expression in the CEP group. No statistically significance correlation was noted between Bcl-2 and P53 and Bcl-2 and Bak protein expression in both examined groups. The obtained data suggests that P53 and Bcl-2 protein expression coupled with decreasing Bak expression are associated with apoptosis and proliferation as well as malignant progression in conjunctival and eyelid tumors.

Gap junctional intercellular communication (GJIC) plays a critical role in tissue development and differentiation and probably in carcinogenesis. The purpose of the study was to evaluate the expression and localisation of Cx43 in 40 cases of mammary dysplasia and 29 cases of breast cancer (without primary chemotherapy). The tissue sections were investigated for Cx43 expression by immunohistochemistry. In the normal mammary gland there was an intercellular, punctate staining pattern, mainly between myoepithelial cells, characteristic of functional gap junctions. In dysplasias there was mainly mixed (cytoplasmic and intercellular) staining and in most cases of breast cancer we observed diffuse or granular, but cytoplasmic, staining of Cx43. Our results demonstrated that expression of Cx43 in dysplasias and breast cancer is changed and GJIC is probably impaired because of disruption of functional gap junction formation especially between breast cancer cells.

Numerous laboratory studies and some epidemiological data have suggested the involvement of the insulin-like growth factor-I receptor (IGF-IR) in breast cancer development and progression. However, data on IGF-IR expression in human tissues, including breast cancer sections, are limited and often inconsistent. We therefore examined by immunohistochemistry the expression of IGF-IR in primary tumors and breast cancer metastases to lymph nodes, and correlated IGF-IR positivity with estrogen receptor (ER) status and selected clinicopathological features. We found that 1) IGF-IR was expressed in primary tumors as well as in lymph node metastases, but the expression in primary tumors was more frequent (56 % vs. 44.4 %); 2) IGF-IR expression in primary tumors was associated with negative node status (p < 0.033); 3) in node-negative primary tumors, IGF-IR positively correlated with ERbeta (p < 0.008; r = 0.538), but not with ERalpha, tumor size or grade; 4) both IGF-IR-positive and IGF-IR-negative primary tumors were found to produce IGF-IR-positive as well as IGF-IR-negative metastases; 5) in metastases, IGF-IR expression did not associate with ERalpha, ERbeta or any of the studied pathobiological markers. The results suggest that IGF-IR could become a viable pharmaceutical target in breast cancer therapy, but such therapy should be based on IGF-IR assessment in primary tumor and metastasis in each potential patient.