Sialidase gene transfection enhances epidermal growth factor receptor activity in an epidermoid carcinoma cell line, A431

Glycosphingolipids expressed in cancer cells have been implicated in the modulation of tumor cell growth through their interaction with transmembrane signaling molecules such as growth factor receptors. For glycosphingolipids to interact with growth factor receptors, the presence of sialic acid seems to be essential. Stable transfection of a gene encoding a soluble Mr 42,000 sialidase into a human epidermoid carcinoma cell line (A431) provided an approach by which the level of terminal lipid-bound sialic acid on the cell surface could be altered. In the sialidase-positive clones, the level of ganglioside GM3 was diminished, and little change was observed in protein sialylation. Sialidase-transfected cells grew faster than control cells. Sialidase expression did not modify the binding of epidermal growth factor (EGF) to its receptor but enhanced EGF receptor (EGFR) tyrosine autophosphorylation as compared to that of parental cells or cells transfected with the vector (pcDNA3) alone. Moreover, the phosphorylation of the EGFR, as well as other protein substrates, was observed at low EGF concentrations, suggesting an increase in the receptor kinase sensitivity. These data provided evidence that changes in ganglioside expression in cancer cells by appropriate gene transfection can dramatically affect EGFR kinase activity. Hence, the modulation of ganglioside expression may represent an approach to alter tumor cell growth.     

Amino-terminal charge affects the periplasmic accumulation of recombinant heregulin/EGF hybrids exported using the Escherichia coli alkaline phosphatase signal sequence

An Escherichia coli expression system that exploits the bacterial alkaline phosphatase (PhoA) signal sequence to translocate recombinant human epidermal growth factor (hEGF) to the periplasm was used to evaluate how changes in the composition and sequence of amino acids near the PhoA-hEGF junction influence the periplasmic accumulation of recombinant protein. A series of chimeric structural genes was generated by in vitro replacement of hEGF sequence with analogous segments from the EGF-like domain of human heregulin (HRG), significantly altering the electrostatic character of the amino-terminal region of the mature protein. Quantitation of HRG/EGF protein in E. coli periplasmic extracts, by RP-HPLC, showed a fourfold decrease after one of two acidic residues located in the amino-terminal region of the mature hEGF, near the PhoA junction, was replaced. An additional threefold decrease was observed when the second acidic residue was replaced with a positively charged lysine. Further extension of the amino-terminal HRG sequence, beyond the first six residues, resulted in net neutralization of a more distant EGF acidic residue with no additional effect on protein yield. The importance of having a negatively charged group in the amino-terminal region of the mature protein was confirmed when insertion of an aspartic acid near the amino-terminus of two poorly expressed hybrid protein sequences resulted in a five- to eightfold increase in their recovery from the periplasm. This study demonstrates the importance of having negatively charged residues near the fusion junction of recombinant proteins expressed in E. coli using the PhoA signal sequence for protein export.     

Tyrosine kinase inhibitors. 14. Structure-activity relationships for methylamino-substituted derivatives of 4-[(3-bromophenyl)amino]-6-(methylamino)-pyrido[3,4-d]pyrimidine (PD 158780), a potent and specific inhibitor of the tyrosine kinase activity of receptors for the EGF family of growth factors

The 4-[(3-bromophenyl)amino]pyrido[3,4-d]pyrimidine PD 158780 is a very potent in vitro inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) (IC50 0.08 nM), and other members of the erbB family, by competitive binding at the ATP site of these signal transduction enzymes. A series of analogues of PD 158780 bearing solubilizing functions off the 6-methylamino substituent were prepared by reaction of the 6-fluoro derivatives with appropriate amine nucleophiles. These were evaluated for their ability to inhibit the tyrosine phosphorylating action of EGF-stimulated full-length EGFR enzyme and for inhibition of autophosphorylation of the EGFR in A431 human epidermoid carcinoma cells in culture. The most effective analogues were those bearing weakly basic substituents through a secondary amine linkage, which proved water-soluble (> 10 mM) and potent (IC50S generally < 1 nM). No clear SAR could be discerned for these compounds with respect to amine base strength or the distance of the cationic center from the chromophore, suggesting that 6-substituents are in a favorable area of bulk tolerance in the enzyme binding site. More distinct SAR emerged for the ability of the compounds to inhibit EGFR autophosphorylation in A431 cells, where analogues bearing lipophilic weak bases were preferred. Representative analogues were evaluated for antitumor effectiveness against four in vivo tumor models. Significant in vivo activity was observed in estrogen-dependent MCF-7 breast and A431 epidermoid tumors. Marginal activity was seen in an EGFR-transfected tumor model, suggesting that while this cell line requires EGF for clone formation in soft agar, other growth factors may be able to replace EGF in vivo. Also, no activity was seen against the SK-OV-3 ovarian cancer model, which is known to express other EGF receptor family members (although it is not clear whether these are absolutely required for growth in vivo). While substantial growth delays were seen in A431 and MCF-7 tumor models, the treated tumors remained approximately the same size throughout therapy, suggesting that the compounds are cytostatic rather than cytotoxic under these test conditions. It remains to be determined if more prolonged therapy has cytotoxic effects in vivo, resulting in net tumor cell kill.     

A search in the genome of Saccharomyces cerevisiae for genes regulated via stress response elements

The importance of a cluster of conserved aromatic residues of human epidermal growth factor (hEGF) to the receptor binding epitope is suggested by the interaction of His10 and Tyr13 of the A-loop with Tyr22 and Tyr29 of the N-terminal beta-sheet to form a hydrophobic surface on the hEGF protein. Indeed, Tyr13 has previously been shown to contribute a hydrophobic determinant to receptor binding. The roles of His10, Tyr22 and Tyr29 were investigated by structure-function analysis of hEGF mutant analogues containing individual replacements of each residue. Substitutions with aromatic residues or a leucine at position 10 retained receptor affinities and agonist activities similar to wild-type indicating that an aromatic residue is not essential. Variants with polar, charged or aliphatic substitutions altered in size and/or hydrophobicity exhibited reduced binding and agonist activities. 1-Dimensional 1H NMR spectra of high, moderate and low-affinity analogues at position 10 suggested only minor alterations in hEGF native structure. In contrast, a variety of replacements were tolerated at position 22 or 29 indicating that neither aromaticity nor hydrophobicity of Tyr22 and Tyr29 is required for receptor binding. CD spectra of mutant analogues at position 22 or 29 indicated a correlation between loss of receptor affinity and alterations in hEGF structure. The results indicate that similar to Tyr13, His10 of hEGF contributes hydrophobicity to the receptor binding epitope, whereas Tyr22 and Tyr29 do not appear to be directly involved in receptor interactions. The latter conclusion, together with previous studies, suggests that hydrophobic residues on only one face of the N-terminal beta-sheet of hEGF are important in receptor recognition.     

In vivo pharmacology and anti-tumour evaluation of the tyrphostin tyrosine kinase inhibitor RG13022

Amplification and increased expression of many growth factor receptors, including the epidermal growth factor receptor (EGFR), has been observed in human tumours. One therapeutic strategy for overcoming EGF autocrine control of tumour growth is inhibition of EGFR protein tyrosine kinase (PTK). A series of low molecular weight molecules have been identified which inhibit the EGFR PTK in vitro and demonstrate antiproliferative activity against human cancer cell lines with high expression of EGFR. A significant growth delay in squamous cancer xenografts has been reported for one of these compounds, the tyrphostin RG13022. Based on these encouraging results, we sought to confirm the activity of RG13022 in vivo and relate the effects to the in vivo plasma disposition. RG13022 and three additional peaks were detected by HPLC following intraperitoneal administration of 20 mg kg-1 RG13022 in MF1 nu/nu mice. RG13022 demonstrated rapid biexponential elimination from plasma with a terminal half-life of 50.4 min. RG13022 plasma concentrations were less than 1 microM by 20 min post injection. A primary product was identified as the geometrical isomer (E)-RG13022. Both RG13022 and its geometrical isomer inhibited DNA synthesis in HN5 cells after a 24 h in vitro incubation (IC50 = 11 microM and 38 microM respectively). Neither RG13022 nor its geometrical isomer displayed significant cytotoxicity. RG13022 had no influence on the growth of HN5 tumours when administered chronically, starting either on the day of tumour inoculation or after establishment of tumour xenografts. The rapid in vivo elimination of RG13022 has potential significance to the development of this and other related tyrphostin tyrosine kinase inhibitors, as plasma concentrations fell below that required for in vitro activity by 20 min post injection. The lack of in vivo tumour growth delay suggests that a more optimal administration schedule for RG13022 would include more frequent injections or continuous administration. An improved formulation for RG13022 is therefore required before further development of this or other similar protein tyrosine kinase inhibitors can be made. Alternative strategies should also be sought which display longer lasting in vivo exposures.     

Pharmacokinetic and pharmacodynamic changes of furosemide after intravenous and oral administration to rats with alloxan-induced diabetes mellitus

We previously developed the "immunogene" approach toward cancer gene therapy using epidermal growth factor receptor (EGFR)-mediated endocytosis. Here, we describe an improved immunogene system, in which the antigen-binding (Fab) fragments of the monoclonal antibody (Ab) B4G7 against the human EGFR were conjugated with poly-L-lysine to form a gene delivery vehicle (designated Fab "immunoporter"). Within 12 hours, the beta-galactosidase beta-gal) gene was transferred via the Fab immunoporter to virtually all of the nuclei of human squamous carcinoma A431 cells that overproduce the EGFR, and the beta-gal enzyme activity was detected within 24 hours and retained for more than 3 days. The beta-gal gene was not transferred into human and mouse cells that were deficient in EGFRs, but it was delivered if those mouse cells were transformed with human EGFR genes. Beta-gal gene transfer via the Fab immunoporter was inhibited by pretreatment with excess amounts of the Fab fragment. The transfer efficiency of the beta-gal gene to A431 cells via the Fab immunoporter was approximately 2%, which is as high as the lipofection method and 20- to 100-fold higher than the whole Ab immunoporter. The transfer of the herpes simplex virus thymidine kinase gene into A431 tumor cells as a form of the thymidine kinase/Fab immunogene was successful, and subsequent treatment with ganciclovir induced remarkable suicide effects which conferred 1000-fold higher drug sensitivity. Thus, the Fab immunogene was substantially improved with regard to the whole Ab immunogene and could be used as a potent gene transfer vehicle for the in vivo targeting of EGFR-hyperproducing tumor cells.     

Enediyne-lexitropsin DNA-targeted anticancer agents. Physicochemical and cytotoxic properties in human neoplastic cells in vitro, and intracellular distribution

Two series of hybrids of a dynemicin A model and DNA minor groove binding lexitropsins were synthesized and their cytotoxic activities were investigated in a panel of human normal and malignant cell lines using a colorimetric assay. Adriamycin was used as a control. Several of the agents demonstrated cytotoxic activity, the extent of which varied with tumor type. IC50s of the hybrids ranged from approximately 14-48 microM following 96 h incubation in the presence of test compound. Intracellular distribution studies were facilitated through endogenous fluorescence of the compounds. Evidence of nuclear uptake of the hybrid agents was demonstrated by confocal laser scanning microscopy. The results warrant further development of DNA-targeted enediyne-lexitropsin hybrids as potential anticancer agents.     

Galactose oxidase-glucan binding domain fusion proteins as targeting inhibitors of dental plaque bacteria

In order to inhibit the growth of bacteria present in the human oral cavity, a novel system which targets antimicrobial agents to dental plaque has been developed. This system involves a hybrid protein consisting of a peptide expressing the bactericidal properties of galactose oxidase (GAO) fused to the glucan binding domain (GBD) of the Streptococcus mutans glucosyltransferase-S enzyme. A gene encoding GAO from the fungus Fusarium sp. has been inserted into an Escherichia coli expression vector and fused to sequences encoding the GBD, which binds to the glucans synthesized by oral streptococci. Bacterial extracts expressing the hybrid protein were tested for their ability to target the GAO activity to an in vitro plaque model consisting of streptococcal cells bound to microtiter plate wells. The binding of the hybrid protein to the streptococcal cells through its GBD and the dependence of binding on the production of glucans by bacteria were demonstrated. Furthermore, killing of three different species of oral streptococci by bound hybrid protein in conjunction with the galactose-lactoperoxidase-iodide cytotoxic system has been demonstrated. These results suggest a novel strategy for controlling dental plaque formation as well as dental caries in humans.     

Topology and function of the Na+/proline transporter of Escherichia coli, a member of the Na+/solute cotransporter family

Penidiamide, a new tripetide containing dehydrotryptamine, glycine and anthranilic acid linked together by two amide bonds, and oxindole were isolated from submerged cultures of Penicillium sp. 62-92. Both compounds preferentially inhibited human synovial phospholipase A2, penidiamide with an IC50 of 30 microM and oxindole of 380 microM. With the exception of U 937 cells (leukemia, human), no cytotoxic activities were detected against HL-60- (leukemia, human), HeLa S3- (epitheloid carcinoma, human), BHK 21- (kidney fibroblasts, hamster), and L1210-cells (leukemia, mouse). No antimicrobial activity was detected for oxindole, and only weak antibacterial activity for penidiamide. The structure of penidiamide was elucidated by spectroscopic methods.     

Distribution of epidermal growth factor receptor and ligands during bronchiolar epithelial repair from naphthalene-induced Clara cell injury in the mouse

Clara cells are primary targets for metabolically activated pulmonary toxicants because they contain an abundance of the cytochrome P450 monooxygenases required for generation of toxic metabolites. The factors that regulate bronchiolar regeneration after Clara cell injury are not known. Previous studies of naphthalene-induced bronchiolar injury and repair in the mouse have shown that epithelial cell proliferation is maximal 1 to 2 days after injury and complete 4 days after injury. Proliferation is followed by epithelial re-differentiation (4 to 14 days). In this study, mice were treated with the environmental pollutant naphthalene to induce massive Clara cell injury. The distribution and abundance of three growth-regulatory peptides (epidermal growth factor receptor (EGFR), epidermal growth factor (EGF), and transforming growth factor (TGF)-alpha) was determined immunochemically during repair of this acute bronchiolar injury. EGFR and its ligands were detected at low levels in cells throughout the lung including peribronchiolar interstitial cells, blood vessels, and conducting airway epithelium. Immediately after naphthalene injury (1 to 2 days), EGFR, EGF, and TGF-alpha are expressed in increased abundance in squamous epithelial cells of the injury target zone, distal bronchioles. These immunopositive squamous cells are detected in clumps in the distal bronchioles at the time when cell proliferation is maximal. EGFR protein expression is decreased slightly 4 to 7 days after injury and continues to decrease below control levels of abundance 14 to 21 days after injury. This down-regulation of EGFR is not reflected in a corresponding decrease in EGF and TGF-alpha protein expression, indicating that control of cell proliferation is regulated at the receptor level. Co-localization of EGFR and bromodeoxyuridine-positive proliferating cells in the same bronchiole indicates that EGFR is up-regulated within the proliferative microenvironment as well as in specific proliferating cells within the injury target zone. The coincident localization within terminal bronchioles of EGFR, EGF, and TGF-alpha to groups of squamous epithelial cells 2 days after naphthalene injury suggests that these peptides are important in up-regulating cell proliferation after Clara cell injury in the mouse.     

Identification of the minimal requirements for binding to the human epidermal growth factor (EGF) receptor using chimeras of human EGF and an EGF repeat of Drosophila Notch

Many proteins contain so-called epidermal growth factor (EGF)-like domains that share the characteristic spacing of cysteines and glycines with members of the EGF family. They are, however, functionally unrelated, despite the fact that the three-dimensional structure of these EGF-like domains, also, is often very similar to that of the EGF receptor agonists. In the present study, we linked an EGF-like repeat from the Drosophila Notch protein to the N- and C-terminal linear tail sequences of human EGF (hEGF), and we showed that this chimera (E1N6E) is unable to bind or activate the hEGF receptor. This recombinant protein was then used as a basic construct for identifying the minimal requirements for high affinity EGF receptor binding and activation. We selectively reintroduced a limited number of important hEGF-derived residues, and by using this unique approach, we were able to make hEGF/Notch chimeras that, compared with wild type hEGF, showed nearly 100% binding affinity and mitogenic activity on HER-14 cells expressing the hEGF receptor.     

Inhibition of growth of primary human tumour cell cultures by a 4-anilinoquinazoline inhibitor of the epidermal growth factor receptor family of tyrosine kinases

The epidermal growth factor receptor (EGFR) is thought to mediate the action of the mitogens EGF and tumour growth factor-alpha (TGF-alpha) in a variety of cancers, including those of the lung, breast and ovary. A number of new selective inhibitors of EGFR tyrosine kinase have now been developed as potential new antitumour agents. We used a potent inhibitor of this tyrosine kinase, 6-amino-4-[(3-bromophenyl)amino]-7-(methylamino)quinazoline (SN 25531; PD 156273), to determine the responses of primary cultures derived from patients with cancer of the lung, ovary, breast, cervix and endometrium. Cells were cultured in 96-well plates and proliferation assessed by incorporation of 3H-thymidine. Measured growth inhibitory concentrations IC50 values) varied from 1 nM to 14 microM with a 1000-fold differential between sensitive and resistant cultures. Results were compared with rates of proliferation, estimated using a paclitaxel-based method. We also measured the IC50 values for the tyrosine kinase inhibitor using a number of established human cell lines, and compared them with EGFR content using fluorescent antibody staining and flow cytometry. The presence of EGFR was found to be necessary, but not sufficient, for in vitro response. Only a small number of cell lines (3 of 7 for lung, 1 of 7 for ovarian, 2 of 3 squamous cell and 0 of 12 for melanoma) were sensitive to the tyrosine kinase inhibitor. In contrast, 40 of the 50 primary cultures (including 14 of 15 lung cancer samples and 14 of 19 ovarian cancer samples) were sensitive.     

Variations in nucleolar organizer regions during 9-10 dimethyl1-2 benzanthracene induced tongue carcinogenesis in rats

BACKGROUND: Many studies have reported the increased expression of epidermal growth factor receptor (EGFR) in various human malignancies and its association with the biologic behavior of the tumors. METHODS: We performed an immunohistochemical analysis of the EGFR in 217 cases of human esophageal squamous cell carcinoma, 161 lymph node metastases and 23 foci of squamous dysplasias. The findings were correlated with clinicopathologic features, including the clinical outcome. Southern blot analysis was performed in 42 cases for the detection of DNA amplification of the EGFR gene and subsequently was correlated with EGFR immunoreactivity. RESULTS: Epidermal growth factor receptor overexpression was detected in 71% of primary tumors and 88% of lymph node metastases, as compared to nonpathologic adjacent esophageal epithelium. Statistically significant correlations were observed between EGFR overexpression and sex, age, histologic type, and the presence of invasion. Tumor staining was classified into two patterns, homogeneous and heterogeneous, based on the distribution of EGFR-positive cells. The immunostaining patterns of primary tumors had a statistically significant correlation with histologic type, the presence of adventitial invasion, histologic stage and lymph node metastasis. There was a tendency toward a worse prognosis for those patients with EGFR overexpression in the primary tumor. Greater than 90% of the foci of squamous dysplasia demonstrated homogeneous EGFR overexpression. DNA amplification of the EGFR was observed in 21% of primary tumors, and all demonstrated immunohistochemical overexpression. CONCLUSIONS: Immunohistochemical overexpression of the EGFR, which was more frequent than EGFR DNA amplification, appears to play an important role in biologic behavior of human esophageal squamous cell carcinomas.     

Inhibition of human squamous cell carcinoma growth in vivo by epidermal growth factor receptor antisense RNA transcribed from the U6 promoter

BACKGROUND: Squamous cell carcinomas of the head and neck (SCCHN), unlike normal mucosal squamous epithelial cells, overexpress epidermal growth factor receptor (EGFR) messenger RNA and protein. EGFR protein is required to sustain the proliferation of SCCHN cells in vitro. To determine whether EGFR expression contributes to tumor growth, we investigated the effect of suppressing EGFR expression in tumor xenografts through in situ expression of antisense oligonucleotides. METHODS: Intratumoral cationic liposome-mediated gene transfer was used to deliver plasmids capable of expressing sense or antisense EGFR sequences into human head and neck tumors, which were grown as subcutaneous xenografts in nude mice. The oligonucleotides were expressed under the control of the U6 RNA promoter. RESULTS: Direct inoculation of the EGFR antisense (but not the corresponding sense) plasmid construct into established SCCHN xenografts resulted in inhibition of tumor growth, suppression of EGFR protein expression, and an increased rate of apoptosis (programmed cell death). Sustained antitumor effects were observed for up to 2 weeks after the treatments were discontinued. CONCLUSION: These results suggest that interference with EGFR expression, using an antisense-based gene therapy approach, may be an effective means of treating EGFR-overexpressing tumors, including SCCHN.     

A constitutively active epidermal growth factor receptor cooperates with disruption of G1 cell-cycle arrest pathways to induce glioma-like lesions in mice

The epidermal growth factor receptor (EGFR) gene is amplified or mutated in 30%-50% of human gliobastoma multiforme (GBM). These mutations are associated usually with deletions of the INK4a-ARF locus, which encodes two gene products (p16(INK4a) and p19(ARF)) involved in cell-cycle arrest and apoptosis. We have investigated the role of EGFR mutation in gliomagenesis, using avian retroviral vectors to transfer a mutant EGFR gene to glial precursors and astrocytes in transgenic mice expressing tv-a, a gene encoding the retrovirus receptor. TVA, under control of brain cell type-specific promoters. We demonstrate that expression of a constitutively active, mutant form of EGFR in cells in the glial lineage can induce lesions with many similarities to human gliomas. These lesions occur more frequently with gene transfer to mice expressing tv-a from the progenitor-specific nestin promoter than to mice expressing tv-a from the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, suggesting that tumors arise more efficiently from immature cells in the glial lineage. Furthermore, EGFR-induced gliomagenesis appears to require additional mutations in genes encoding proteins involved in cell-cycle arrest pathways. We have produced these combinations by simultaneously infecting tv-a transgenic mice with vectors carrying cdk4 and EGFR or by infecting tv-a transgenic mice bearing a disrupted INK4a-ARF locus with the EGFR-carrying vector alone. Moreover, EGFR-induced gliomagenesis does not occur in conjunction with p53 deficiency, unless the mice are also infected with a vector carrying cdk4. The gliomagenic combinations of genetic lesions required in mice are similar to those found in human gliomas.     

Bcl-2 overexpression is associated with resistance to paclitaxel, but not gemcitabine, in multiple myeloma cells

Multiple myeloma (MM) is an incurable disease despite an initial response-rate of >60% with conventional or high-dose chemotherapy using glucocorticosteroids (i.e. dexamethasone), or alkylating agents (i.e. melphalan). Although these agents are capable of inducing complete remission (CR) in >50% of MM patients, resistance develops rapidly, in >90% of patients, within 2 years of treatment. Therefore, there is a need for new drugs for the treatment of relapsing and refractory MM patients. Gemcitabine (GEM) is a pyrimidine analog that blocks DNA synthesis, whereas, paclitaxel (TAX) is a mitotic spindle poison that promotes microtubular aggregation. Since it appears that these two drugs have different cellular targets, we examined the effect of each drug individually for several parameters and for possible synergy. We studied the cytotoxic effect of TAX and GEM on MM cells expressing varying levels of the antiapoptotic protein bcl-2, which is overexpressed in the majority of myeloma cell from MM patients. We found that both drugs are cytotoxic by inducing apoptosis, however, the extent of apoptosis with TAX, but not with GEM was dependent on the levels of bcl-2 expression. We further investigated the effect of TAX and GEM on the cell cycle distribution and on the levels of bcl-2. The results indicate that the two drugs have different modes of action with respect to each parameter tested. TAX induced arrest of the cells in the G2/M phase of the cell cycle, regardless of bcl-2 levels, however, apoptosis was induced in mitotic cells expressing relatively low levels of bcl-2. In contrast, GEM caused apoptosis of cells in the S-phase, regardless of level of bcl-2 expression. A major difference between TAX and GEM was in their effects on the levels of bcl-2. Whereas, TAX induced an early downregulation of bcl-2 (only in the cells with relatively low levels of bcl-2), treatment with GEM did not affect bcl-2 levels. The effects of TAX on both the cell cycle and bcl-2 were detected very early (4-8 h) and preceded the onset of apoptosis. GEM and TAX act synergistically, at low doses (IC50 of 0.5 microM for GEM and 0.025 microM for TAX), to effectively kill bcl-2 overexpressing cells that are resistant to higher doses (0.25 microM) of TAX alone. Therefore, we have initiated a phase II clinical trial of TAX and GEM for MM patients refractory to current therapy.     

Radiologic features of carcinomas arising in hiatal hernias

The plant toxin saporin is a ribosome-inactivating protein which inhibits protein synthesis and growth of both normal and tumour cells. Its cytotoxic activity can be increased by coupling with antibodies recognizing cell surface antigens. In this work we performed experiments to test the hypothesis that saporin induces cell death via apoptosis. Exposure to saporin induced apoptosis in different cellular models, such as human peripheral blood B lymphocytes and neutrophils, in the Daudi B-cell line, and in the haemopoietic cell lines HL-60 and TF-1. This was indicated by: (a) the appearance of typical morphological features such as chromatin condensation, nuclear fragmentation and blebbing of plasma membranes: (b) DNA degradation into oligonucleosomal fragments: (c) the appearance of apoptotic cells on DNA flow cytometry as a cell population with reduced DNA content (A0 region). The fraction of cells showing features of apoptosis ranged from 19 +/- 5% for TF-1 cells to 35 +/- 8% for neutrophils. In experiments with normal peripheral blood B lymphocytes or with Daudi cells, we compared the activity of native saporin with that of an immunotoxin hybrid molecule obtained by binding the toxin to two bispecific antibodies recognizing both saporin and the B lymphocyte-specific antigen CD22 (Sap/BsAb complexes). Saporin bound to the antibodies was 2-3 logs more effective than native saporin in inducing apoptosis, with maximal inhibitions being observed at concentrations of 10(-6) M for native saporin and 10(-9)-10(-8) M for the hybrid molecules. These findings indicate that treatment with saporin results in apoptosis of target cells and suggest that this may be relevant to the therapeutic use of saporin-containing immunotoxins. In fact, if used in vivo as an immunotoxin, its cytotoxic activity could be devoid of more extensive and non-specific tissue damaging effects as would be the case if saporin induced necrosis of target cells.     

5-hydroxytryptamine1A receptor-mediated effects on adenylate cyclase and nitric oxide synthase activities in rat ventral prostate

AIM: To investigate the patterns of expression of transforming growth factor alpha (TGF-alpha) and epidermal growth factor receptor (EGFR) in squamous metaplasia and squamous cell carcinomas of the urinary bladder with and without schistosomiasis. METHODS: Immunohistochemical study of the expression of TGF-alpha and EGFR in squamous metaplasias (n = 12) and various grades of squamous cell carcinomas (n = 21) of the bladder with and without schistosomiasis. RESULTS: Focal cytoplasmic and membranous positivity for EGFR and TGF-alpha was seen in all cases of squamous metaplasia. The markers were diffusely coexpressed in a concordant pattern in areas of hyperplastic keratinising squamous metaplasia. A similar pattern of positivity was seen in verrucous carcinomas (n = 2) and well differentiated squamous carcinomas (n = 6). Progressive loss of differentiation was associated with increasing loss of EGFR staining while TGF-alpha staining was retained. Squamous cell carcinoma in situ (n = 2) showed focal positivity for TGF-alpha and EGFR. There were no differences in staining patterns between cases with and without schistosomiasis. CONCLUSIONS: The coexpression of TGF-alpha and EGFR by well differentiated squamous cell carcinomas and hyperplastic keratinising squamous metaplasia is consistent with the active regulatory role exerted by this autocrine loop. There is regional absence of expression of EGFR but not of TGF-alpha in squamous cell carcinomas of lesser differentiation, suggesting heterogeneity of such control in these tumours. The focal expression of the two markers in squamous cell carcinomas in situ indicates a possible second pathway of oncogenesis for less differentiated tumours. These observations may have important implications for the effectiveness of putative growth factor based treatments.     

Five new bioactive sesquiterpenes from the fungus Radulomyces confluens (Fr.) Christ

The three protoilludanes radulone A (1), radulone B (2) and radudiol (3), the illudalane radulactone (4) and the illudane radulol (5) were isolated from the extracts of the culture fluids of the basidiomycete Radulomyces confluens. The structures of the five new compounds were determined by spectroscopic techniques. Radulone A (1) is a potent inhibitor of human and bovine platelet aggregation stimulated by different agonists, inhibiting preferentially the aggregation of human platelets induced by ADP with an IC50 value of 2 microM. In addition 1 exhibits cytotoxic and antimicrobial activities. The other four compounds exhibited weak antimicrobial and cytotoxic activity.