Ascorbyl stearate inhibits cell proliferation and tumor growth in human ovarian carcinoma cells by targeting the PI3K/AKT pathway.

Ascorbyl stearate is a lipophilic, vitamin C derivative with antitumorigenic properties. The molecular mechanism(s) underlying the anticarcinogenic effect of this compound have not been well documented. The effect of ascorbyl stearate was studied in a panel of human ovarian epithelial cancer cells. Treatment with ascorbyl stearate caused a dose-dependent inhibition of the cell proliferation. The antiproliferative effect was due to the arrest of cells in the S/G2-M-phase of the cell cycle. Treatment of OVCAR-3 cells with ascorbyl stearate also inhibited PI3K/AKT activity. The presence of a constitutively active AKT protected OVCAR-3 cells from the effects of ascorbyl stearate, suggesting that this nutraceutical targets the PI3K/AKT pathway. The administration of ascorbyl stearate by gavage induced involution of human ovarian carcinoma xenografts in nude mice. These studies indicate that the antiproliferative effect of ascorbyl stearate on ovarian epithelial cancer cells is associated with decreased PI3K/AKT activity, and point toward the PI3K/AKT signaling pathway as a target for this drug.

Up-regulation of VEGF expression and related neo-angiogenesis in childhood high-grade gliomas: implications for anti-angiogenic anti-neoplastic therapy.

Vascular endothelial growth factor (VEGF) is a homodimeric, disulfide-linked glycoprotein which exhibits endothelial cell-specific mitogenic properties. VEGF is also a potent inducer of vascular permeability. There is considerable experimental evidence that VEGF isoforms are strongly involved in provoking neoangiogenesis of neoplastic cells and, consequently, the growth and progression of primary neoplasms (i.e., astrocytic gliomas), including the formation of an invasive and metastatic immunophenotype (IP). During this immunohistochemical study, the presence and tissue localization of VEGF121 was observed in anaplastic, high-grade astrocytomas (AAs) and in glioblastoma multiforme (GBMs) employing the specific monoclonal antibody against it. A sensitive, four-step, alkaline phosphatase-conjugated antigen detection technique was used. The immunoreactivity demonstrated a cytoplasmic, cell surface and extracellular matrix localization pattern in more than 90% of the tumor cells, with high intensity immunoreactivity (++++, A,B) in every high-grade astrocytic glioma tissue. VEGF121 expression was identified mostly within the cytoplasm of tumor cells, suggesting an embryonic, undifferentiated and more malignant cellular IP of high-grade gliomas. Tumor-related neo-angiogenesis and endothelial cell proliferation were also present. The great majority of high-grade astrocytic gliomas are incurable with the three classic therapeutic modalities. In the future, the development of targeted anti-neoplastic treatment strategies, adapted to individual patients, will require molecular identification of the different classes of neoplasm (including subtypes of astrocytomas) according to their stages, biology, prognosis and therapeutic options.

Cyclooxygenase-2 (COX-2) overexpression in childhood brain tumors.

The overexpression of COX enzymes has been demonstrated in human neoplasms at various sites, including the colon, gastrointestinal tract, lung, skin and recently in brain tumors. In this study, COX-2 receptor overexpression in primary childhood brain tumors was determined and the distribution pattern of COX-2 receptors was examined. A sensitive, 4-step, alkaline phosphatase conjugated antigen detection technique was used and a specific monoclonal antibody for medulloblastomas/ primitive neuroectodermal tumors (MEDs/PNETs), anaplastic, high-grade astrocytomas (ASTRs) and in glioblastoma multiformes (GMs) was employed. All of the 14 MEDs/PNETs observed demonstrated high levels of immunoreactivity (overexpression), with the highest immunostaining intensity (grades A and B). However, of the 14 subtypes of astrocytic tumors examined, the COX-2 receptor expression level did not even approach those of the MEDs/PNETs levels. However, significant differences were found when comparing low grade pilocytic ASTRs to high grade anaplastic ASTRs and glioblastomas. In two low grade pilocytic ASTRs, the expression level never exceeded 20%, while in high grade glial tumors (6 anaplastic ASTRs and 6 GMs) 30 to 50% of the tumor cells overexpressed COX-2 receptors, documenting an increase in COX-2 receptor overexpression with the increasing grade of the astrocytic tumor. In view of these findings, it would appear likely that COX-2 inhibitors may represent a chemo-preventive tool in treating childhood brain tumors, which are the leading cause of solid tumor cancer death in children under the age of 20.

Differentiation-dependent expression and mitogenic action of interleukin-6 in human colon carcinoma cells: relevance for tumour progression.

Interleukin (IL)-6 mRNA expression in general is low in normal, adenomatous and cancerous human colon mucosa, except in rather undifferentiated lesions, in which IL-6 is overexpressed. Cytokeratin (CK) 8-positive carcinoma cells were identified by double immunostaining as almost exclusive source of IL-6. Likewise, in five (sub)clones of primary culture COGA-1 and COGA-13 human colon carcinoma cells, and in three established cell lines (Caco-2/AQ, Caco-2/15 and HT-29), efficient translation of IL-6 mRNA into protein was observed only in the least differentiated COGA-13 cells. Notably, IL-1beta (5 ng/ml) enhanced IL-6 release in COGA-13 cultures by three orders of magnitude. Although all cell clones studied expressed the IL-6 receptor (IL-6R), rhIL-6 (1-100 ng/ml) had a significant effect on cellular proliferation only in highly differentiated Caco-2 cells. Our data imply that IL-6, when released from rather undifferentiated carcinoma cells, particularly in response to IL-1beta, can advance tumour progression through paracrine growth stimulation of normal or highly differentiated colon tumour cells with intact STAT-3-mediated IL-6 signalling.

Epidermal growth factor receptor (EGFR) expression in childhood brain tumors.

Overactivation of epidermal growth factor receptor (EGFR) signaling has been recognized as an important step in the pathogenesis and progression of multiple forms of cancer of epithelial origin. Reports regarding EGFR family members in brain tumors are sparse and, thus, the significance of EGFR expression in childhood brain tumors is unclear. In this study, the expression of the EGFR family members was analyzed in 22 medulloblastomas. During the immunohistochemical study, a sensitive, four-step, alkaline phosphatase conjugated antigen detection technique was employed. The results demonstrated the presence of c-erbB-2 (HER-2) and c-erbB-4 (HER-4) in 10 to 50% of the neoplastic cells of high-grade glial tumors with high immunoreactivity, while c-erbB-3 (HER-3) was only detected in less than 10% of the neoplastically-transformed cells. In a follow-up, 70% of children, usually under 4 years of age, with c-erbB-2 (HER-2)-positive MEDs/PNETs, succumbed to the cancer. The Kaplan-Meier estimation revealed a significant correlation between c-erbB-2 expression and survival (p = 0.002), suggesting that c-erbB-2 (HER-2) is probably a prognostic marker for limited survival. Medulloblastoma is the most common malignant brain tumor that occurs during childhood. Multimodality treatment regimens have substantially improved survival in this disease; however, the tumor is incurable in about one-third of patients with medulloblastoma, and the current treatment has a detrimental effect on long-term survivors. As such, the results of this study further support the idea that targeting EGFR alone, or in combination with its downstream mediators, represents a promising new approach for the management of childhood brain tumors. Moreover, c-erbB-2 (HER-2) expression may also be of use in better classifying brain tumors.

Immunocytochemical detection of members of the caspase cascade of apoptosis in childhood medulloblastomas.

During the process of programmed cell death (PCD), the cell disintegrates into small, membrane-bound apoptotic bodies. Caspase-3 is ubiquitously expressed in normal and neoplastically-transformed human cells and serves as an executioner in the apoptotic or PCD pathway. During our immunocytochemical study, a sensitive, four-step, alkaline phosphatase-conjugated antigen detection technique was employed. The results demonstrated the presence of apoptotic activity within the cellular microenvironment of childhood medulloblastoma/primitive neuroectodermal tumor. The observations identified the cytoplasmic presence of caspase-3 in more than 20% of neoplastic cells. The immunocytochemical expression pattern demonstrated a translocation tendency from the cytoplasm to the cell nuclei in the apoptotic cells in about 5% of the tumor cells. Caspase-3 presence was also detected in the tumor infiltrating lymphocytes (TILs), representing the host's immune, mostly CD8+, cytotoxic, tumor-associated antigen (TAA)-directed effector cells. This phenomenon may play an important role in these tumors' maintenance of immune privilege and evasion of immune attacks. We suggest that the grade and intensity of apoptosis may not only have diagnostic and prognostic significance, but could also play a leading role in the biological (fourth modality) antineoplastic treatment of these highly malignant, neuroectodermal brain tumors.

Flat and polypoid adenocarcinomas of the colorectum: A comparative histomorphologic analysis of 47 cases.

"Flat" colorectal adenomas and adenocarcinomas are well documented in the Japanese literature but only sporadically reported in the English literature. The present study involved systematic morphological analysis of a large series of colorectal carcinomas (CRCs) to determine the frequency of these "flat" CRCs (FCRCs) and analyze their pathological characteristics and associated patient survival. The study group comprised 47 patients (19 females and 28 males) with primary CRC who underwent colorectal resection at the H. Lee Moffitt Cancer Center between 1997 and 2002. These cases were selected based on the gross appearance of the tumors and after review of all of the hematoxylin and eosin-stained tumor sections in a series of 190 consecutive colorectal resections for CRCs. Application of strict morphological criteria classified 22 tumors as FCRCs. For comparison, 25 "polypoid" CRCs (PCRCs) were also identified. Cases of ulcerative fungating annular CRCs and CRCs with mixed gross appearance were excluded from this analysis. Clinicopathologic data, including patient survival, were compared for FCRCS and PCRCs. Statistical analyses were carried out using the chi(2) or Fisher's exact test and log-rank tests. Overall, 22 of 190 CRCs (11%) were found to meet the morphological criteria of FCRCs. Mean patient age was 70.6 years (range, 55 to 87) for FCRCs versus 68.5 years (range, 54 to 91) for PCRCs, and mean tumor size was 4.7 cm (range, 1.6 to 9) for FCRCs versus 4.4 cm (range, 0.5 to 10) for PCRCs. None of the 22 FCRCs and only 1 of 25 (4%) PCRCs were well differentiated; 17 of 22 (77%) FCRCs and 23 of 25 (92%) PCRCs were moderately differentiated; and 5 of 22 (22%) FCRCs and 1 of 25 (4%) PCRCs were poorly differentiated (P = 0.0087). FCRC cases were staged as 0 stage T1, 3 (14%) stage T2, and 19 (86%) stage T3; PCRC cases, as 4 (16%) stage T1, 14 (56%) stage T2, and 7 (28%) stage 3 (P = 0.000031). Similarly, angiolymphatic invasion was identified in 12 of 22 (54%) FCRCs versus 4 of 25 (16%) PCRCs (P = 0.0123). Although some differences between FCRCs and PCRCs were observed on resection in terms of nodal status (N), presence of metastases (M), and perineural invasion, these differences were not statistically significant. In comparison with PCRCs, FCRCs were associated with significantly shorter postresection patient survival at 1 to 5 years (P = 0.028). We have demonstrated in this report that a proportion of primary CRCs resected at our institution were indeed "flat." Furthermore, these FCRCs exhibited higher histological grades, higher T stage, more frequent angiolymphatic invasion, and shorter patient survival compared with PCRCs. Based on these data, FCRC appears to be a worse subtype of colon cancer than PCRC. Further appraisal of FCRCs and additional studies to further elucidate the molecular mechanisms underlying their morphogenesis are warranted.

Cyclooxygenase-2 expression in right- and left-sided colon cancer: a rationale for optimization of cyclooxygenase-2 inhibitor therapy.

Cyclooxygenase-2 (COX-2) has been identified as a potential target for prevention and therapy of human colorectal cancers (CRC) and other cancers. Because right-sided colon cancers (RSCCs) exhibit clinicopathologic and genetic differences from left-sided colorectal cancers (LSCRCs), determination of COX-2 status in these subsets of CRCs may be clinically relevant in designing COX-2 inhibitor trials for CRC and in subsequent assessment of objective therapeutic response to such therapy. Thirty-six primary CRC resection specimens (18 left, 18 right) from 36 patients were evaluated. Representative tumor sections were subjected to immunohistochemical analysis of COX-2. A semiquantitative system was used to score cytoplasmic COX-2 immunostaining. The tumors were considered COX-2 positive if more than 10% tumor cells showed COX-2 staining. Clinicopathologic and COX-2 data were compared for LSCRCs versus RSCCs. All 18 LSCRCs and 13 of 18 (72%) RSCCs were well to moderately differentiated. Overall rates of COX-2 positivity for the LSCRCs versus RSCCs were 67% (12 of 18) and 33% (6 of 18), respectively (P = 0.04). Furthermore, 11 of 12 (92%) COX-2 positive LSCRCs and 3 of 6 (50%) COX-2 positive RSCCs were stage II-IV at resection. All 12 COX-2 positive LSCRCs were associated with advanced primary tumor and 4 of 12 (33%) LCRCs had distant metastases. These associations could not be evaluated for the RSCCs because of the limited number of COX-2 positive cases. The more frequent expression of COX-2 in LSCRCs as compared with RSCCs supports the hypothesis that COX-2 expression may be related to genetic alterations specific to right- or left-sided CRCs. Further studies are needed to elucidate such relationships. Our data also suggest that stratification of patients with CRC into right- and left-sided subsets may be important in optimal patient selection for COX-2 inhibitor therapy and for subsequent assessment of objective therapeutic response.

Insulin-like growth factor 1 receptor activates c-SRC and modifies transformation and motility of colon cancer in vitro.

Colorectal carcinomas have been found to express increased levels of IGF1 and IGF1-R, as compared to normal or adenomatous colonic mucosa, and it has been postulated that a subset of colorectal cancers are under the autocrine regulation of the IGF1/IGF1-R system. In this study, we selected human colorectal carcinoma cell lines with high (SW620, HT29, L4A) and low (CaCo2, and HCT 116) expression of IGF1-R by flow cytometry. Compared to the IGF1-R(-) cells, the IGF1-R(+) cells revealed a more aggressive phenotype as demonstrated by a higher proliferation rate (approximately 2-fold increase) in response to IGF1, higher degree of transformation (approximately 5-to-15-fold increase in colony formation in soft agar), increased resistance to serum deprivation-induced apoptosis [1-7 apoptotic cells/5 microscopic fields, as compared to 37 to 101 apoptotic cells/5 microscopic fields of the IGF1-R(-) cells], and higher migratory capability measured by a wounding assay [IGF1-R(+) cells migrated a distance of up to 15 millimeters from the cut edge of the monolayer, while the IGF1-R(-) cells were able to migrate only 2-3 millimeters away from the same reference point]. While the cell lines overexpressing the IGF1-R had higher levels of Src activation, the use of a Src inhibitor reduced the IGF1-R protein expression, slowed down the proliferation of IGF1-R(+) cells, and reduced their colony formation in soft agar. Based on the above observations, we conclude that an overexpressed and activated IGF1-R may increase the degree of transformation and motility of colon cancer cells by activating c-Src.

Antigen presentation by dendritic cells and their significance in antineoplastic immunotherapy.

Dendritic cells (DCs) are present in essentially every mammalian tissue, where they operate at the interface of innate and acquired immunity by recognizing pathogens and presenting pathogen-derived peptides to T lymphocytes. According to the research group of Shortman, experimental results suggest a "dual" DC differentiation model, demonstrating the existence of both myeloid-derived (with characteristic IF: CD11b+, CD11c+, CD8alpha- and DEC205+) and lymphoid-derived DCs (showing CD11b- CD11c-, CD8alpha+ and DEC205+ IF). DCs, including interdigitating cells (IDCs) and Langerhans cells (LCs), are characterized by dendritic morphology, high migratory mobility and are the most effective, "professional" cells for antigen presentation in primary immune responses. Most of the DCs express immunocytochemically detectable antigens like: S-100, CD1a, CD40 receptor, adhesion molecules (ICAM-1 or CD54, LFA-1 and LFA-3), integrins (CD11a, CD11c and CD18), CD45, CD54, co-stimulatory molecules (B7-1 or CD80, B7-2 or CD86), F418, MHC class I and II and DEC-205, multilectin receptor, immunostimulatory cytokine (IL-12) and, of course, Fc and complement receptors. Following recognition and uptake of antigens, mature dendritic cells (DCs) migrate to the T lymphocyte rich area of draining lymph nodes, display an array of antigen-derived peptides on the surface of major histocompatibility complex (MHC) molecules and acquire the cellular specialization to select and activate naive antigen-specific T lymphocytes. Immunotherapeutic ideas are based on the ability of the mammalian immune system to recognize neoplastically transformed cells. Immunotherapy of human neoplasms has always represented a very attractive fourth-modality therapeutic approach, especially in light of the many shortcomings of conventional surgical, radiation and chemotherapies in the management of neoplastically transformed cells. The cancer vaccine approach to therapy is based on the notion that the immune system could possibly mount a rejection strength response against the neoplastic cell conglomerate. The efficiency of DCs for T lymphocyte stimulation moved a number of research groups to develop DC- based immunotherapy approaches. The failure of cancer vaccines may be attributed to the relationship between host and neoplasm: through a natural selection process, the host facilitates the selective enrichment of clones with highly aggressive neoplastically transformed cells, being in various stages of differentiation and only during certain stages express neoplastic cell specific molecules.

Restoration of the thymic cellular microenvironment following autologous bone marrow transplantation.

Mammalian thymic histogenesis can be morphologically divided into three consecutive stages: 1) epithelial; 2) lymphopoietic or lympho-epithelial; and 3) differentiated cellular microenvironmental, with formation of Hassall's bodies (HBs). The marked reduction of the thymic cellular microenvironment (TCM) is a well-controlled physiological process and is presumably under both local and global regulation by the cells of the RE meshwork and by the neuroendocrine axis, respectively. In humans, the age-related decline of facteur thymique sérique (FTS) levels in blood begins after 20 years of age and FTS completely disappears between the 5th and 6th decade of life. In contrast, serum levels of thymosin-alpha 1 and thymopoietin seem to decline earlier, starting as early as 10 years of age. The influences of other hormones on the thymic involution have also been characterized: testosterone, estrogen and hydrocortisone treatment results in marked involution, cortisone and progesterone administration causes slight to moderate, while use of desoxycorticosterone has no effect. Since the thymus is the primary T-lymphopoietic organ during mammalian ontogenesis, its age-related involution with the typical immunomorphological alterations can be held responsible only for a decline in antigen-specific T-lymphocyte immune functions. Thymic involution and diminished T-lymphocyte proliferation can be partially restored by thymic tissue transplantation or administration of thymic hormones. The stimulus for thymic cell proliferation and differentiation is genetically determined within the organ implant. The only partial reconstitution of CD4+ T-helper-lymphocyte subset after anti-neoplastic chemotherapy and autologous BTM represents a significant, therapy-complicating, clinical problem. After high-dose chemotherapy, the restoration of thymus-dependent CD4+ T-lymphocyte genesis was reported only in children. Our radiation, stem cell transplantation, and hormone treatment experiments in animals resulted in age- and time-dependent regeneration of the cytoarchitecture of the TCM, as well as intrathymic lymphopoiesis.

Bacteria-related spontaneous and therapeutic remission of human malignancies.

The development of specific immunotherapeutic approaches to cancer treatment is making progress. However, a crucial clinical break-through in cancer vaccination is still missing. Spontaneous clinical remissions of malignancies are a rare phenomenon. The mechanisms are not clear but there are remissions described in correlation with bacterial infections. The use of bacteria or bacterial components might be a promising path for non-specific immunotherapy of tumors and might be a helpful adjuvant for specific immunotherapy. Here, we briefly review spontaneous remissions of various malignancies in humans, the impact of bacterial infection on tumor regression and the utilization of bacterial components in clinical trials.

MAGE-1, a cancer/testis-antigen, expression in childhood astrocytomas as an indicator of tumor progression.

During the last decade, the aberrant expression of normal testicular proteins in neoplastically transformed cells became common knowledge. Cancer/testis-antigens (CTAs) represent a novel family of immunogenic proteins. The MAGE genes were initially analyzed from melanomas and turned out to have an almost exclusively neoplasm-specific expression pattern. This expression pattern might contribute to the genetic instability of neoplastically transformed cells. In normal adult tissues, most 23 human MAGE genes are expressed only in the testis, but only in the mitotic spermatogonia (germ cells) and in the primary spermatocytes. The immunocytochemistry was carried out on routine, formalin-fixed, paraffin-wax-embedded 3 to 4 mm thick astrocytoma (ASTR) tissue sections. A four step, indirect, biotin-streptavidin based method was employed with alkaline phosphatase enzyme conjugation. Immunocytochemical presence and cellular localization of the MAGE-1 CT-antigen, employing anti-MAGE-1 MoAB was observed only in anaplastic, high-grade ASTRs (100%), certainly including glioblastomas, in this study. The immunoreactivity was always heterogeneous, showing a cytoplasmic pattern and loosely grouped cells with similar staining characteristics being detected within the cellular and hormonal microenvironment of the ASTRs. We never identified MAGE-1, CT-antigen expression in the lowest grade, pilocytic ASTRs. The MAGE-1 CTA expression levels may also be used to evaluate the malignant and dedifferentiation tendencies of low-grade ASTRs and predict the likelihood of mutations of the genome and further dedifferentiation towards even more malignant anaplastic ASTR and glioblastoma multiforme IPs.

Survivin expression in childhood medulloblastomas: a possible diagnostic and prognostic marker.

Survivin is a member of the inhibitor of apoptosis gene family that is expressed in embryonic tissues during human ontogenesis and most human malignancies, but it is not present in the majority of normal adult tissues. Survivin is also a chromosomal passenger protein required for physiological cell divison. Survivin blocks apoptosis, via its BIR domain, by either directly or indirectly blocking the function of the members of the caspase cascade. The expression of this apoptosis inhibitor protein in medulloblastomas (MEDs) was examined for the first time. During the immunohistochemical study, a sensitive, four-step, alkaline phosphatase conjugated antigen detection technique was employed. The results did, in fact, demonstrate the presence of survivin in 10 to 50 per cent of medulloblastoma (MED) cells with medium intensity immunoreactivity (++, B) in this neuroectodermal brain tumor. These results indicate that survivin is probably not only a diagnostic marker, but also an important prognostic marker for MEDs/PNETs and may be useful in the future grading of malignancy in MEDs, much as grading is done today for astrocytomas (ASTRs). Furthermore, the almost exclusive neoplastic expression of survivin will allow development of new antineoplastic, immunotherapeutic strategies.

Vaccines for immunotherapy of breast cancer and prostate cancer: new developments and comparative aspects.

This review summarizes the most recent findings and the newest approaches in the design of vaccines for breast cancer and prostate cancer. The antigens associated with breast cancer and prostate cancer and their use in vaccine development are discussed. We consider the comparative aspects in the new vaccines and focus on their clinical potential. The future directions in designing cancer vaccines are discussed.

Racial differences in childhood tumors.

The purpose of this brief review is to inform the reader of the appearance of childhood neoplasms along racial and ethnic lines. The Lag time, general aspects and specific tumors, such as astrocytomas, medulloblastomas and those of the skin, including cases affecting the face and mouth, are discussed. Other tumors discussed are melanomas, those of the nasopharynx, liver, bone, Hodgkin's disease and especially leukemias as primary forms. Special emphasis was put on the racial and other variabilities producing various combinations and specific responses to treatment.

Role of p53, CD44V6 and CD57 in differentiating between benign and malignant follicular neoplasms of the thyroid.

The distinction between follicular adenoma (FAD) and follicular carcinoma (FCA) of the thyroid can be particularly challenging in routine practice of diagnostic surgical pathology. It often requires examination of several histologic sections in order to identify the presence of unequivocal capsular and/or vascular invasion. To investigate the role of immunohistochemical markers in the differential diagnosis of follicular lesions of the thyroid, we studied the pattern of expression of p53, Bcl-2 and of the adhesion molecules CD44V6 and CD57, in 20 FADs and 21 FCAs of the thyroid. Ninety percent of FCAs exhibited strong nuclear p53 expression. p53 stain was seen in only 15% of FADs (p<0.0001), and it was weak. Bcl-2 cytoplasmic immunoreactivity was observed in 57% of FCAs and 60% of FADs (p=1.00). Similarly, membranous CD44V6 staining was seen in 81% of FCAs, but in only 20% of FADs (p=0.0001). CD57 was present in the cytoplasm of 71% of FCAs and 15% of FADs (p=0.0004). None of the markers studied correlated with tumor size. The results of this study indicate that immunohistochemical detection of p53, CD44V6 and CD57 may have practical utility in the differential diagnosis of FCAs from FADs in routine surgical pathology.

Immunocytochemical detection of members of the caspase cascade of apoptosis in high-grade astrocytomas.

During the physiological process of PCD, the cell initiates a sequence of events culminating in the disintegration of the cell into small, membrane-bound apoptotic bodies. The intrinsic part of the PCD program arises from the mitochondria when it releases cytochrome c from the mitochondrial intermembrane space into the cytosol, forming the caspase-activating complex or apoptosome. The family of caspases is involved in the execution of genetically controlled PCD. Caspase-3 is expressed in normal and neoplastically transformed human cells and, like other caspases, is synthesized as an inactive, 32kDa proenzyme. Caspase-6 cleaves nuclear mitotic apparatus protein (NuMA) and mediates the shrinkage and fragmentation of cell nuclei. Caspase-8 is an initiation caspase that activates the caspase cascade during apoptosis, while caspase-9 is the initiator caspase in the caspase cascade in apoptotic normal and neoplastically transformed cells. During our immunocytochemical study, a sensitive, four-step, alkaline phosphatase conjugated antigen detection technique was employed. The results did in fact demonstrate the presence of high apoptotic activity within the cellular microenvironment of high-grade astrocytomas and glioblastomas. The observations identified cytoplasmic expression of caspase-3 and caspase-6 in more than 50 per cent of tumor cells, caspase-8 and caspase-9 in more than 10 per cent of tumor cells in high-grade anaplastic ASTR and glioblastoma. The immunocytochemical expression pattern in about 10 per cent of the tumor cells for caspase-3 and caspase-6 and about 1 to 5 per cent of the tumor cells for caspase-8 and caspase-9 demonstrated a translocation tendency from the cytoplasm to the cell nuclei in the apoptotic cells. This phenomenon may play an important role in these tumors' maintenance of immune privilege and evasion of immune attacks. We suggest that caspase-3, -6, -8 and -9 immunocytochemistry could have prognostic and immunotherapeutic significance in the treatment of these highly malignant glial tumors.

Vascular stroma-derived endothelial colony forming progenitor cells adapt to tumour growth.

Under appropriate nutrient agar culture conditions, primary or xenografted human and animal tumour biopsy-derived cell suspensions will form two types of colony. The first type, consisting of tight colonies of round cells which form tumours when introduced into nude mice, is of neoplastic origin. The second type of colony, the cells of which fail to form tumours on injection into nude mice, consists of loose colonies of larger, inter-connecting elongated bi- or tripolar cells and is thought to originate from vascular stroma-derived endothelial colony forming progenitor cells (V-ECPC). The likely importance of V-ECPC to tumour growth is emphasised by a positive correlation between the VECPC-derived endothelial cell colonies and both tumour vascularity and growth rate. A high cloning efficiency obtained from tumours of particularly intense vascular nature indicates that this cell is of importance in vascular adaptation and therefore tumour growth. In contrast, avascular, fibrotic tumour tissue yielded very low numbers of stromal vascular endothelial cell colonies. The results suggest that stromal vascular endothelial cell colonies do not arise from the mature fibroblastic elements of the tumour stroma, but rather from cells within actively growing regions. Tritiated thymidine uptake studies show that the vascular stroma-derived endothelial colony forming progenitor cells cell are cycling. Cell separation studies have characterized the as yet morphologically unidentified V-ECPC as having a sedimentation rate of 4.7 mm./hr and a mean density of 1.068 g/cm3 and hence a calculated volume of 450 microns 3.

Glutathione-S-transferase pi expression and activity is increased in colonic neoplasia.

Glutathione-S-transferase (GST) isoenzymes are involved in the conjugation of glutathione to electrophilic carcinogens. Recent studies have shown increased levels and activities of GST in different tumors suggesting their role in carcinogen detoxification. This study compared GST activity levels and GST-pi protein expression in paired samples of colorectal cancer, adenoma and adjacent normal mucosa from a total of thirteen patients. GST was isolated from human colorectal specimens and assayed spectrophotometrically; Western immunoblot analysis was used to quantify GST-pi levels. GST activity was greater in both colorectal cancer and adenomas than in adjacent normal colonic tissue, although statistical significance was achieved only when comparing colorectal cancer to normal tissue. Based on these observations, we conclude that increased GST activity may be a useful marker of colonic neoplasia.