Altered expression of cell cycle regulatory proteins in benign and malignant bone and soft tissue neoplasms.

BACKGROUND: The binding of cyclins to cyclin-dependent kinases regulates cell proliferation. Overexpression of cyclins is believed to deregulate the cell cycle in human tumors. Here the expression of G1 cyclins D1 and D3, and of Ki-67 in a variety of bone and soft tissue sarcomas was assessed as compared to adjacent normal tissue and to a subset of leiomyomas. MATERIALS AND METHODS: Twenty-nine human bone and soft tissue sarcomas were evaluated. Tissue sections from each case were subjected to immunostaining for cyclin D1, cyclin D3 and Ki-67 using the avidin-biotin complex method. RESULTS: Cyclin D1 nuclear positivity was detected in 28% of sarcomas and in none of the leiomyomas. Cyclin D3 nuclear positivity was present in 62% of sarcomas and in none of the leiomyomas. Ki-67 nuclear staining was positive in 86% of sarcomas but in only 16% of leiomyomas. In addition, upregulation of cyclin D1 was observed in leiomyosarcomas, pleomorphic sarcomas and gastrointestinal stromal tumors, but not in liposarcomas or osteosarcomas. Cyclin D3, however, was expressed in all of the sarcoma types including 2 out of 5 liposarcomas and 1 out of 4 osteosarcomas. The normal soft tissue adjacent to the tumors when present (10 cases) was negative for cyclin D1 and D3, and expressed Ki-67 in 5% of the cell nuclei. The expression of cyclin D3 was also noted in human sarcoma cell lines (SKLMS, MG63, SaOS-2 and HT1080) by Western blot. CONCLUSION: The higher expression of cyclin D1 and D3 and of Ki-67 in bone and soft tissue sarcomas, as compared to leiomyomas and peritumoral normal soft tissue, suggests that high cyclin expression may contribute to deregulation of the cell cycle in bone and soft tissue tumors. These data suggest a role of cyclins in the process of human sarcomagenesis.

Cyclooxygenase-2 (COX-2) expression in human endometrial carcinoma and precursor lesions and its possible use in cancer chemoprevention and therapy.

In recent years, the design of new antineoplastic agents that can halt the progression of human malignancies with minimal systemic damage has been at the forefront of cancer research, with cyclooxygenase-2 (COX-2) as a major target molecule. With an aim to demonstrate the expression and role of COX-2, the principal putative target of COX-2 inhibitor therapy, in endometrial adenocarcinoma (EACA) and precursor lesions, atypical complex hyperplasia (ACH) and endometrial hyperplasia (EH), an immunohistochemical (IHC) analysis of 22 primary human EACAs and 14 precursor lesions was carried out. Relevant clinicopathological data were tabulated from a random computer-generated sample of 22 primary EACA patients, treated by hysterectomy at our institution. Representative tumor sections including adjacent precursor lesions and normal endometrium (NE) were immunostained with human monoclonal anti-COX-2. Qualitative and semi-quantitative COX-2 IHC staining scores were determined based on the proportion of immunoreactive cells and the intensity of cytoplasmic COX-2 expression. Fisher's exact test and the Wilcoxon Rank Sum test were used for statistical analysis. Mean patient age was 68 years (range 51-93). All 22 EACAs were of endometrioid type, of which ten (45%) were grade I, eight (36%) grade II and four (18%) were grade III. Overall, four out of nine (44%) EHs, four out of five (80%) ACHs, and 18 out of 22 (88%) EACAs were COX-2 positive. The mean COX-2 IHC scores for EH and EACAs were 33 (SD 24.11) and 76 (SD 54.57), respectively (p = 0.022). Strong or moderate COX-2 expression was observed in 17 out of 22 (77%) adenocarcinomas as compared to two out of 14 (14%) of the precursor lesions (EH and ACH). The areas of adenomyosis were COX-2 positive, while myometrial smooth muscle and normal fallopian tube tissues stained negative for COX-2. The demonstration of frequent and strong expression of COX-2 in human EACAs supports a possible role for COX-2 inhibitors. Furthermore, an increasing expression of COX-2 from EH to invasive EACAs suggests potential usefulness of COX-2 inhibition to halt the progression of precursor lesions to invasive endometrial cancers.

Value of multilevel sectioning for improved detection of micrometastases in sentinel lymph nodes in invasive squamous cell carcinoma of the vulva.

UNLABELLED: Clinical usefulness of sentinel lymph node (SLN) biopsy has been demonstrated in the management of early vulvar cancer. However, what constitutes a negative SLN has not been well defined. Furthermore, to what extent the SLNs should be sectioned for the greatest likelihood of detection of micrometastases and whether multilevel sectioning will further increase this detection rate in this setting have not been well studied. We analyzed 280 groin lymph nodes (SLNs=45, non-sentinel [NSLNs]=235) in 14 patients with invasive squamous cell carcinoma (ISCC) of the vulva treated with vulvectomy and inguinal SLN and NSLN dissection at the H. Lee Moffitt Cancer Center (HLMCC) between 1996 and 2001. Each SNL was evaluated for micrometastases by H&E and pancytokeratin AE1/3 (CKAE1/3) immunohistochemical staining. All negative SNLs (N=40) were sectioned times 3 (x3) at 50-micron intervals and independently reviewed by two pathologists in order to assess the utility of this inexpensive and logical approach to identifying additional micrometastases. Also, the Wilcoxon Rank Sum Test was used to determine if there was an association between tumor size, depth of invasion and SNL status. The patient age ranged from 35 to 81 years (mean 59 yrs); size of invasive tumor from 1.0 to 7.0 cm (mean 3.4 cm); depth of invasion from 3 to 25 mm (mean 10.8 mm). Of 45 SLNs examined from 14 patients, 11% (5/45) SNLs were positive for micrometastases on initial H&E and/or CKAE1/3 stains. Eighty-nine per cent (40/45) SNLs were negative in the remaining 9 patients. None of the latter 40 SNLs showed micrometastases on additional multilevel sectioning. Instead 3 of 135 NSLNs examined in these 9 patients revealed micrometastases on H&E (skip-micrometastases). Mean tumor size (cm) and depth of invasion (cm) were 4.06 (s.d. 1.89) and 1.20 (s.d. 0.35) for SLN (+) and 3.02 (s.d. 2.12) and 1.01 (s.d. 0.86) for SLN (-) tumor subsets (p values 0.385 and 0.348, respectively). CONCLUSION: Following routine H&E and CK AE1/3 stains, multilevel sectioning does not appear to detect additional micrometastases in sentinel lymph nodes in squamous cell carcinoma of the vulva. Even though mean tumor size and depth of invasion were greater in SNL (+) as compared to SLN (-) tumor subsets in our series, this difference did not reach statistical significance.

COX-2, NSAIDs and human neoplasia. Part I: Colorectal neoplasms.

Cyclooxygenase-2 (COX-2), the inducible cyclooxygenase isozyme involved in the conversion of arachidonic acid (AA) to biologically active prostanoids, has become the subject of intense interest during the last few years. The recent surge of interest stems from seminal studies that correlated elevated expression of COX-2 with tumor induction and progression, and epidemiological studies that correlated reduced risk of developing certain types of cancers with chronic use of non-steroidal anti-inflammatory agents (NSAIDs). Although these observations were first reported with colorectal cancer (CRC), similar findings have subsequently been made with other types of cancers. A wide spectrum of studies continue to be undertaken in both laboratory and clinical settings to elucidate the mechanisms underlying these anti-tumor effects of COX-2 for potential translation into cancer chemoprevention and therapy. The aim of this article is to present a review of COX genes, the prostaglandin-cyclooxygenase relationship, the role of COX-2 in carcinogenesis and the rationale for targeting COX-2 with NSAIDs for cancer chemoprevention. Special emphasis is given to the role of COX-2 expression in the genesis and progression of colorectal neoplasia, and its correlation with other pathological characteristics of CRC. Preliminary observations on COX-2 expression in inflammatory bowel disease (IBD)-related colorectal neoplasia are also presented.

Preneoplastic lesions of the prostate-clinical, pathological and molecular biological aspects.

Needle biopsy is the mainstay of definitive diagnosis of prostate cancer (PCA). While prostate-specific antigen (PSA) screening has facilitated early diagnosis of PCA, it has also resulted in an increase in the proportion of prostate biopsies showing various preneoplastic lesions (PNLs). At times such lesions are the sole finding in the limited amount of tissue available for assessment in an individual biopsy. Hence accurate identification of these lesions is important to avoid errors in the diagnosis of prostatic malignancy and in patient management. Furthermore, some interesting observations have been made regarding the molecular biological aspects of various PNLs during the last decade. In parallel with anatomic and physiological differences in various human races, racial differences have also been observed regarding the incidence of prostatic intra-epithelial neoplasia. This review focuses on prostatic intraepithelial neoplasia (PIN), atypical adenomatous hyperplasia (AAH) and atypical prostatic glands or atypical small acinar proliferation (ASAP) as putative preneoplastic lesions of the prostate. These lesions are reviewed with reference to their overall incidence, histopathological findings, histological differential diagnosis, clinical significance and molecular biological aspects.

Expression of proline-directed protein kinase, (p34cdc2/p58cyclin A), a novel cell proliferation marker in childhood brain tumors.

The presence of two proteins of the proline-directed protein kinase (PDPK), the catalytic subunit p34cdc2 and the regulatory subunit p58cyclin A was determined in seven primitive neuroectodermal tumors (PNETs), three choroid plexus neoplasms and eleven astroglial tumors. The highest expression was registered in the cellularly undifferentiated PNETs and glioblastoma multiforme from the astroglial malignant group. Rabbit immunoantiserum against the two subunits of PDPK, a cell proliferation marker, was employed to detect proliferation activity in childhood brain tumors. The PDPK activity was present from Gl- to M-phases in 21 childhood brain tumors with different central nervous system (CNS) localization and cellular atypia. Immunocytochemical analysis employed an indirect, alkaline phosphatase conjugated biotin-streptavidin antigen detection technique on frozen and routine, formalin-fixed and paraffin-wax-embedded tissue sections of brain tumors. We compared the proliferation activity in the cells of normal, morphologically changed and neoplastically transformed choroid plexus. The average proliferation activity was low in comparison with other tissues. The results in normal and neoplastically transformed choroid plexus were very similar. The lowest proliferation activity in the astroglial group belonged to pilocytic ASTRs. The use of cell differentiation as a prognostic factor in primary brain tumors has already been established and is strongly suggested by our research group. Further systematic neoplasm studies and regular employment of these two polyclonal antibodies for immunocytochemical screening experiments are necessary to determine their true diagnostic and prognostic significance.

Effects of butaclamol, clopenthixol, mepromazine and cannabinol stereoisomers on apoptosis induction.

The efflux pump of multidrug resistant mdr cells have different sensitivities to some stereoisomeric forms of CNS-active compounds. The ABC transporters of mdr cells were more sensitive to (-)butaclamol than to its stereoisomeric counterpart (8), which may function to alter the membrane structure. We suppose that the drug-accessible membrane structure possesses an important role in the induction (or prevention) of apoptosis. Therefore the apoptosis-inducing effect of three stereoisomeric pairs was studied on mouse lymphoma cells. In these experiments levo- and dextromepromazine had similiar effects. The cis- and trans-clopenthixol were less effective in apoptosis induction than the 12H-benzo(a)-phenothiazine used as a positive control. The effect of stereoisomeric pairs on induced apoptosis was studied when the cells were exposed to the stereoisomers for 60 minutes before subjection apoptosis induction by benzo(a)phenothiazine, a well-known apoptosis inducer. Then the pretreated cells were exposed to 12H-benzo(a)-phenothiazine for 60 minutes. The samples were washed and incubated for 24 hours. The cells were stained with annexin-V-FITC and propidium iodine and investigated by flow cytometry. The mdr cells with increased membrane integrity may result in the preferential killing of multidrug resistant cancer cells in the presence of some stereoisomers.

Matrix metalloproteinases in neoplasm-induced extracellular matrix remodeling in breast carcinomas.

Structural changes in the extracellular matrix (ECM) are necessary for cell migration during normal and pathologic tissue remodeling and neoplastic cell invasion. The matrix metalloproteinases (MMPs) and their inhibitors have been identified to be critical modulators of ECM composition and are thus, crucial in neoplastic cell progression, invasion and metastasis. Expression of MMP-2, -3, -9, -10, and -13 was investigated in human breast carcinomas (BCs) employing an indirect, biotin-streptavidin based, alkaline phosphatase conjugated immunocytochemical technique. Evaluation of the results was based on (a) the percent of neoplastically transformed cells/surrounding stroma that reacted positively and (b) a measure of staining intensity [graded from A (highest) to D (negative)]. The two forms of stromelysin, MMP-3 and -10, share 82% sequence homology, but exhibit differences in cellular synthesis and inducibility by cytokines and growth factors in vitro. Strong overall expression of MMP-3 and -10 was found in BCs, especially in the ECM adjacent to blood vessels. Positive immunoreactivity could be seen for these two MMPs in the ECM surrounding over 90% of the neoplastically transformed cells (++++), and the staining intensity was also the strongest possible (A). High intensity immunoreactivity (A,B) but focal was detected employing a MoAB targeted against the MMP-9 enzyme. No presence of MMP-2 or -13 could be established in the BC cases observed by us. Based on these results we propose that MMP-3 and -10 are implicated in the pathogenesis of BC, while MMP-9 is possibly involved in neo-angiogenic events also closely associated with growth and expansion of the neoplastically transformed cell mass, as well as metastasis of individual, extremely aggressive, expressing dedifferentiated cellular immunophenotype (IP) cell clones selected during the microevolution of the BC.

Immunocytochemical detection of leukocyte-associated and apoptosis-related antigen expression in childhood brain tumors.

During systematic cell-surface antigen expression profile analyses of 76 primary childhood brain tumors [34 medulloblastomas (MED)/primitive neuroectodermal tumors (PNETs) and 42 astrocytomas (ASTR)], a library of monoclonal antibodies (MoABs) directed against various leukocyte-associated, lymphocyte cell-line differentiation antigens in childhood brain tumors was utilized. The antigens were detected employing an indirect, biotin-streptavidin conjugated alkaline phosphatase (AP) immunocytochemical technique. Major histocompatibility complex (MHC) class I restricted, tumor-associated antigen (TAA) specific, CD8(+) cytotoxic T lymphocytes (CTL) were identified in 58/76 (76.32%) brain tumors, and usually represented 1-10% of all cells, but in some cases 30-44% of the cells were CD8(+). CD4(+), MHC class II restricted helper lymphocytes were present in 65/76 (85.53%) brain tumors, and accounted for 1-10% of the observed cells. Macrophages were present in 74/76 (97.37%) brain tumors, and their number also represented 1-10% of all observed cells in the brain tumor frozen sections. Leukocyte common antigen (LCA) expression was detected in all 76 (100%) brain tumors studied. MoAB UJ 308 detected the presence of premyelocytes and mature granulocytes in 60/76 (78.95%) brain tumors. Natural killer (NK) cells were not defined in the observed brain tumors. The great majority of childhood glial tumors, particularly ASTRs express Fas (APO-1/CD95) receptor whereas normal cells in the central nervous system (CNS) do not. FasR is a transmembrane glycoprotein which belongs to the nerve growth factor/tumor necrosis factor (NGF/TNF) receptor superfamily. As part of our screening, the 42 childhood ASTRs were also investigated for expression of CD95. We detected strong expression (strong intensity of staining, number of stained cells 50-100%) of FasR, employing formalin fixed, paraffin-wax embedded tissue slides. Brain tumors and melanomas have been shown to produce their autocrine FasL, and are even capable of switching CD95-related signal transduction from the PCD pathway to a proliferative pathway. In view of our results, we conclude that: (1) the tumor infiltrating leukocytes in MEDs/PNETs and ASTRs represent a very diverse population and are present in a great majority of the cases studied; (2) the strong expression of FasR in ASTRs provides a manner in which T lymphocytes may exert their anti-tumor effects, but may also represent yet another way that tumors may evade the immune response; and (3) further observations of the expression of various antigens involved in juxtacrine, in situ growth control are necessary for the refinement of cellular immunotherapeutical approaches in the treatment of human malignancies.

Invasion and metastasis: the expression and significance of matrix metalloproteinases in carcinomas of the lung.

Structural changes in the extracellular matrix (ECM) are necessary for cell migration during tissue remodeling and tumor invasion. The matrix metalloproteinases (MMPs) and their inhibitors have been shown to be critical modulators of ECM composition and are thus, crucial in neoplastic cell invasion and metastasis. Expression of MMP-2, -3, -9, -10, and -13 was investigated in human lung adenocarcinomas employing an indirect alkaline phosphatase conjugated immunocytochemical technique. Evaluation of the results was based on (a) the percent of neoplastically transformed cells/surrounding stroma that reacted positively and (b) a measure of staining intensity [graded from A (highest) to D]. The two forms of stromelysin, MMP-3 and -10, share 82% sequence homology, but exhibit differences in cellular synthesis and inducibility by cytokines and growth factors in vitro. Strong overall expression of MMP-3 and -10 was found in lung adenocarcinomas, especially in the ECM adjacent to blood vessels. Positive immunoreactivity could be seen for these two MMPs in the ECM surrounding over 90% of the neoplastically transformed cells (++++), and the staining intensity was also the strongest possible (A,B). Focal (+), high intensity (A,B) staining could be detected for MMP-2, -9, and -13. Thus, it seems that the stromelysins are involved in the generalized growth and expansion of the neoplastic cell mass, while MMP-2, -9 and -13 are involved in the neoangiogenic and focal clonal selection and expansion phenomena associated with in situ tumor progression, invasion of the microvasculature, and metastasis.

Matrix metalloproteinase expression in malignant melanomas: tumor-extracellular matrix interactions in invasion and metastasis.

Structural changes in the extracellular matrix (ECM) are necessary for cell migration during tissue remodeling and tumor invasion. The matrix metalloproteinases (MMPs) and their inhibitors have been shown to be critical modulators of ECM composition and are, thus, crucial in neoplastic cell invasion and metastasis. Expression of MMP-2, -3, -9, -10, and -13 was investigated in human malignant melanomas (MMs) employing an indirect alkaline phosphatase conjugated immunocytochemical technique. Evaluation of the results was based on (a) the percent of neoplastically transformed cells/surrounding stroma that reacted positively and (b) a measure of staining intensity [graded from A (highest) to D]. The two forms of stromelysin, MMP-3 and -10, share 82% sequence homology, but exhibit differences in cellular synthesis and inducibility by cytokines and growth factors in vitro. Strong overall expression of MMP-3 and -10 was found in MMs, especially in the ECM adjacent to blood vessels. Positive immunoreactivity could be seen for these two MMPs in the ECM surrounding over 90% of the neoplastically transformed cells (++++), and the staining intensity was also the strongest possible (A,B). Focal (+), high intensity (A,B) staining could be detected for MMP-2, -9, and -13. Thus, it seems that the stromelysins are involved in the generalized growth and expansion of the neoplastic cell mass, while MMP-2, -9 and -13 are involved in the neoangiogenic and focal clonal selection and expansion phenomena associated with in situ tumor progression, invasion of the microvasculature, and metastasis.

Immunocytochemical detection of matrix metalloproteinase expression in prostate cancer.

Structural changes in the extracellular matrix (ECM) are necessary for cell migration during tissue remodeling and tumor invasion. The matrix metalloproteinases (MMPs) and their inhibitors have been shown to be critical modulators of ECM composition and are, thus, crucial in neoplastic cell invasion and metastasis. Expression of MMP-2, -3, -9, -10, and -13 was investigated in human prostatic carcinomas employing an indirect alkaline phosphatase conjugated immunocytochemical technique. Evaluation of the results was based on (a) the percent of neoplastically transformed cells/surrounding stroma that reacted positively and (b) a measure of staining intensity [graded from A (highest) to D]. The two forms of stromelysin, MMP-3 and -10, share 82% sequence homology, but exhibit differences in cellular synthesis and inducibility by cytokines and growth factors in vitro. Strong overall expression of MMP-3 and -10 was found in lung adenocarcinomas, especially in the ECM adjacent to blood vessels. Positive immunoreactivity could be seen for these two MMPs in the ECM surrounding over 90% of the neoplastically transformed cells (++++), and the staining intensity was also the strongest possible (A,B). Focal (+), low to high intensity (C to A) staining could be detected for MMP-2, while no immunoreactivity was observed employing MoABs directed against MMP-9 and -13. Thus, it seems that the stromelysins are involved in the generalized growth and expansion of the neoplastic cell mass, while MMP-2 is involved in the neoangiogenic and focal clonal selection and expansion phenomena associated with in situ tumor progression, invasion of the microvasculature, and metastasis.

Immunocytochemical detection of the expression of members of the matrix metalloproteinase family in adenocarcinomas of the pancreas.

Structural changes in the extracellular matrix (ECM) are necessary for cell migration during tissue remodeling and tumor invasion. The matrix metalloproteinases (MMPs) and their inhibitors have been shown to be critical modulators of ECM composition and are, thus, crucial in neoplastic cell invasion and metastasis. Expression of MMP-2, -3, -9, -10, and -13 was investigated in human pancreatic adenocarcinomas employing an indirect alkaline phosphatase conjugated immunocytochemical technique. Evaluation of the results was based on (a) the percent of neoplastically transformed cells/surrounding stroma that reacted positively and (b) a measure of staining intensity [graded from A (highest) to D]. The two forms of stromelysin, MMP-3 and -10, share 82% sequence homology, but exhibit differences in cellular synthesis and inducibility by cytokines and growth factors in vitro. Strong overall expression of MMP-3 and -10 was found in lung adenocarcinomas, especially in the ECM adjacent to blood vessels. Positive immunoreactivity could be seen for these two MMPs in the ECM surrounding over 90% of the neoplastically transformed cells (++++), and the staining intensity was also the strongest possible (A,B). Focal (+), high intensity (A,B) staining could be detected for MMP-2 and -13, while no immunoreactivity was observed employing the anti-MMP-9 MoAB. Thus, it seems that the stromelysins are involved in the generalized growth and expansion of the neoplastic cell mass, while MMP-2 and -13 are involved in the neoangiogenic and focal clonal selection and expansion phenomena associated with in situ tumor progression, invasion of the microvasculature, and metastasis.

A model of BAX gene delivery to human lung cancer.

In this report we have investigated the effects of BAX in enhancing apoptosis in two primary non-small cell lung cancer cell lines. A count of the apoptotic cells by TUNEL staining revealed that almost 70% of BAX over-expressing cells died, while very few apoptotic cells were detectable in the wildtype cells or in the cells transfected with an empty vector. These findings suggest that de-regulated expression of BAX may provide a novel mechanism for initiating cell death in non-small cell lung cancer cells. Further studies are needed to better define the involvement of this protein in the complex mechanism of lung carcinogenesis and to definitely demonstrate the therapeutic utility of targeting this pathway.

Sex steroids, biochemical markers, bone mineral density and histomorphometry in male osteoporosis patients.

The present study investigates the role of sex steroids, especially serum estradiol and serum testosterone in male osteoporosis patients and their association to established markers of bone turnover as also to BMD results and histomorphometric findings. Included were patients with secondary osteoporosis due to steroid medication, anticonvulsive medication and alcohol consumption, and heavy smoking patients. 100 males aged from 30 to 78 years were investigated for osteodensitometry (DEXA) and assessment of biochemical bone turnover markers (venous blood samples, 24 hour urine samples). In 40 of these patients bone biopsies were taken for histomorphometry. Laboratory investigations were made for serum Ca, P, parathyroid hormone (PTH), osteocalcin (OC), carboxyterminal extension peptide of type I procollagen (PICP), bone specific alkaline phosphatase (B-ALP), 25OH-vit.D, testosterone, estradiol, gonadotropines, and deoxypyridinoline and hydroxyproline from 24-hour urinary collection. Regions of interest for osteodensitometry with DEXA technique were the lumbar spine L1-L4 and the femoral neck (Ward's triangle). All of the patients examined had low bone mineral density (BMD) values compared to age- and sex-matched controls. Results from descriptive statistics showed hypogonadism in 26.4%, 25 OH-vitamin D deficiency in 26.2% and high serum estradiol in 59.1% of patients, compared to age- and sex-matched controls. 8.5% had elevated PTH levels. Multivariate analysis of data showed no significant correlation between BMD and semiquantitative histomorphometric findings (scaled from 1-5), neither a significant correlation between serum testosterone/estradiol and BMD. A significant correlation was observed between testosterone and estradiol values (r = 0.389, p = 0.008), and between OC and BMD results at ward's triangle (p = 0.008). In steroid treated patients (n = 12) significant differences were found for PTH (P < 0.01), 25 OH-Vit.D (p < 0.05) and urinary deoxypyridinoline (p < 0.05) as compared with the other patient group (n = 88). In summary we found high serum estradiol in 59.1% of our patients collective with low BMD, there was no correlation between BMD and histomorphometric findings. We observe a significant positive correlation between testosterone and estradiol values, but we did not find any association to bone turnover markers or BMD results.

Serum levels of immunoreactive bone sialoprotein in osteoporosis: positive relations to established biochemical parameters of bone turnover.

Bone Sialoprotein (BSP), synthesized by osteoblasts and osteoclasts, is a highly glycosylated and phosphorylated protein, accounting for approximately 5-10% of noncollagenous proteins of bone extracellular matrix. The present study investigates possible correlations between serum values of immunoreactive Bone Sialoprotein in relation to established bone turnover markers like osteocalcin (OC), bone alkaline phosphatase (B-ALP) and the c-terminal extension peptide of type-I-Procollagen (PICP) in 170 osteoporosis patients (female n = 144, male n = 26) in order to evaluate the usefulness of BSP in the diagnosis of bone disease. Fasting venous blood samples were collected from our osteoporosis outpatients in the morning and stored at -80 degrees C until processing. Serum levels of BSP were determined by RIA, OC and B-ALP were measured by IRMA, and PICP was assessed employing an ELISA technique. A significant correlation was found between BSP serum values and B-ALP (r = 0.532, p = 0.0001). Median serum BSP levels were 8.0 micrograms/l, median B-ALP values were 22.39 U/ml in these patients. Also a significant correlation was observed between BSP and OC (r = 0.588, p = 0.0001), more pronounced in the female patient group (r = 0.632, p < 0.0001). A weak association between BSP and PICP in the female group was detected (r = 0.398, p = 0.0001). In the female group BSP was inversely related to serum estradiol levels (r = -0.274, p = 0.002) as to BMD (DEXA) at the lumbar spine and femoral neck. In conclusion, BSP might be a useful marker of non-collagenous organic bone matrix in laboratory assessment of bone turnover, being inversely related to BMD at lumbar spine and femoral neck and showing significant correlations to established markers of bone turnover like B-ALP and OC.

A permanent cell line of the crayfish Orconectes limosus as a potential model in comparative oncology.

A cell line, designated OLGA-PH-J/92, was established from neuronal tissue of the crayfish Orconectes limosus. To date the cell line has been subcultured more than 150 times. From the original cell line two 'daughter' cell lines and one cloned cell line were isolated. Best growth was obtained when the cells were incubated in Eagle's Minimum Essential Medium supplemented with 6% fetal bovine serum at a temperature of 27 degrees C. Under these conditions the population doubling time lasted between 23 and 25 hours. The shape of the cells is dendritic, but can change to spherical when conditions are less optimal. The cell lines showed features of transformation, such as anchorage independence, loss of contact inhibition, and low serum requirement. The number of chromosomes found in the cell lines ranged from 11 to 136, while in the donor species numbered between 98 and 106. It will be of interest to study if the O. limosus cell lines grow malignantly in vivo. OLGA-PH-J/92 and the derivative cell lines should also be suitable for studying viral infections in crustaceans.

Matrix metalloproteinase expression in childhood astrocytomas.

Structural changes in the extracellular matrix (ECM) are necessary for cell migration during tissue remodeling and local neoplastic cell invasion. The matrix metalloproteinases (MMPs) and their inhibitors have been shown to be critical modulators of ECM composition and are thus, crucial in tumor cell invasion and metastasis. The immunocytochemical profile of MMP-2, -3, -9, -10, and -13 expression was observed in 24 primary human childhood astrocytomas (ASTRs) employing an indirect alkaline phosphatase conjugated antigen detection technique. Evaluation of the results was based on (a) the percent of neoplastically transformed cells that reacted positively and (b) a measure of staining intensity [graded from A (highest) to D (negative)]. The two forms of stromelysin, MMP-3 and -10, share 82% sequence homology, but exhibit differences in cellular synthesis and inducibility by cytokines and growth factors in vitro. Strong overall expression of MMP-3 and -10 was found in ASTRs, especially in the ECM adjacent to blood vessels. Positive immunoreactivity could be seen for these two MMPs in the ECM surrounding over 90% of the neoplastically transformed cells (+4) and the staining intensity was also the strongest possible (A,B). No immunoreactivity was detected using antibodies directed against MMP-2, -9, and -13. Based on these results, MMP-3 and -10 are implicated in the pathogenesis of pediatric ASTRs. Further characterization of the expression and utilization of MMPs and their inhibitors in the progression of ASTRs may establish differential regulation and utilization of the various MMPs during the progression of glial tumors, from low-grade pilocytic ASTR to high-grade glioblastoma multiforme.

Immunocytochemical detection of the homeobox B3, B4, and C6 gene products in breast carcinomas.

Breast cancer (BC) represents the most frequent neoplasm in women with a risk of incidence between 10% and 12%. The detection of tumor associated and oncofetal antigen re-expression in a variety of neoplastically transformed cell types has aided in the more precise diagnosis and prognostication of human cancers. The homeobox (HOX) genes encode proteins which contain a 61 amino acid DNA-binding homeodomain and are involved in the transcriptional regulation of other genes during normal onto- and histogenesis. The class I HOX genes are organized in four clusters on different chromosomes in humans, with a high conservation in the order of the genes within each of these clusters. Re-expression of HOX gene products has been reported in a wide variety of neoplastically transformed cells and it seems quite likely that the HOX genes represent yet another class of oncofetal antigens involved in both normal development and carcinogenesis, as well as tumor progression. The expression pattern of three HOX gene products (HOX-B3, -B4, and -C6) was examined immunocytochemically in 11 human breast carcinoma (BC) tissues. In all observed BC cases, HOX-C6 was present in over 90% of the neoplastically transformed cells (+4) demonstrating a high grade (A and B) staining intensity. The same expression pattern was defined for the other two observed proteins (HOX-B3 and -B4; over 90% or +4 and a high grade staining intensity or A and B). Current treatment of BC encompasses the three "classic" modalities of therapy: surgical resection, radiotherapy, and chemotherapy. Although advances have been made, we still face great difficulties in the treatment of this deadly human neoplasm. Therefore, we are always seeking novel tumor associated antigens (TAAs), including oncofetal antigens, to use as molecular targets in cancer cell directed fourth modality immunotherapy.

Expression of p21 in non small cell lung cancer relationship with PCNA.

The first cyclin dependent kinase inhibitor to be discovered was the p21 cdk interacting protein (a.k.a., WAF1, Cip1, CAP20, Sdi1, mda6). p21 expression may or may not be dependent on p53. This pathway also inhibits DNA replication by merit of p21's interaction with PCNA, but it has also been shown that this same inhibitory interaction with p21 does not affect PCNA DNA repair abilities. We assessed the immunohistochemical expression of p21 protein in 60 curative surgical resected non small cell lung cancers relating it to the expression of PCNA to clarify the contribution of the p21/PCNA pathway to the development of NSCLC. We did not find any relationship between PCNA and p21 expression. This last result may indicate that the mechanism by which PCNA controls the DNA repair is the most important activity of this protein during lung cancer progression and development, compared to its contribution to cell proliferation. In fact, this last event is strongly counteracted by p21 expression, which in this last case works as an inhibitor of PCNA expression. In conclusion this study highlighted the important role of the p21/PCNA pathway in lung carcinogenesis, pointing out the contribution of PCNA to the response to lung aggression and not only it's role as a proliferation index. Therefore, these results offer a background to further study to evaluate potential novel therapeutic approaches to lung cancer treatment.