p53-Independent tumorigenic progression of human prostate cells.

We have previously described the development of radiation transformed human fetal prostate epithelial cells, 267B1. Using this in vitro model system, we investigated the molecular mechanisms of prostate carcinogenic progression by comparing nontumorigenic (267B1/B) and tumorigenic (267B1/D) cells. We examined the G1- to S-phase transition in synchronized cells to determine if the progression of 267B1 cells from nontumorigenic to tumorigenic was the consequence of a perturbation in the G1- to S-phase transition involving p53, pRb, p21, or p16. Nontumorigenic 267B1/B cells showed a time-dependent increase in the expression of p53 and a corresponding increase in p21 following exposure to ionizing radiation (6 Gy). The levels of pRb and p16 protein were virtually unchanged. In contrast, tumorigenic 267B1/D cells exhibited a p53-independent induction of p21 protein with a parallel increase in p16 protein in response to ionizing radiation, but no change in pRb was observed. These results suggest that the progression of 267B1 cells from nontumorigenic to tumorigenic involves p53-independent processes.

Rapamycin-induced apoptosis is p53-independent in human prostate carcinoma PC-3 cells.

We have investigated the mechanisms of rapamycin-induced growth inhibition and apoptosis in the PC-3 prostate carcinoma cell line. Rapamycin induced apoptosis as well as the expression of p21(waf1) mRNA and protein, independent of p53. Rapamycin treatment also resulted in: a decrease in cdk2 kinase activity; an increase in hypophosphorylated retinoblastoma protein (pRb); a dephosphorylation of p70 S6 kinase; and, growth-arrest in G(1)-phase of cell cycle. These data suggest that rapamycin-induced growth arrest and apoptosis occur through the p53-independent induction of p21(waf1). Since this induction occurred soon after rapamycin treatment, possibly, the early induction of p21(waf1) and G(1)-arrest are important components of the mechanism by which rapamycin induces apoptosis in PC-3 cells.

Cell cycle-specific patterns of ADP-ribosylation and the effect of ionizing radiation.

The enzyme poly(ADP-ribose)polymerase (PARP) has been implicated in several cellular processes by the demonstration of ADP-ribosylation of proteins which play a role in cellular repair of DNA damage, response to ionizing radiation and apoptosis. In addition, ADP-ribosylation has been shown to play a role in cell cycle progression. To investigate the role of ADP-ribosylation in cell cycle progression, we have synchronized human squamous carcinoma cells (SQ-20B) and examined the changes in the pattern of ADP-ribosylation of proteins through the stages of the cell cycle. Moreover, synchronized SQ-20B cells were irradiated to determine the effects of irradiation on the patterns of ADP-ribosylation through the cell cycle phases. These studies showed the ADP-ribosylation of 3 specific proteins (70, 68 and 40 kDa) to change through the cell cycle. Furthermore, ionizing radiation altered the ADP-ribosylation patterns of these 3 proteins. Using a double antibody method, we determined that cyclins A, B1 and D1, cdc2 and PCNA are not modified by ADP-ribosylation.

Modification of nucleolar protein B23 after exposure to ionizing radiation.

The responses of cells to ionizing radiation include induction and/or suppression of the expression of genes and proteins. In our investigations of alterations in cellular protein expression in response to ionizing radiation, we have used the techniques of two-dimensional polyacrylamide gel electrophoresis and silver staining. We compared the nuclear protein profiles of control and irradiated (6 Gy, 4 h postirradiation) radioresistant squamous carcinoma cells (SQ-20B) and observed an alteration in the expression of a 40 kDa protein: control nuclei express a protein isoform with pI values between 5.4-5.8, while irradiated nuclei express a more acidic variant with pI values between 5.2-5.5. Using the cyanogen bromide/O-phthalaldehyde method followed by microsequencing analysis, we obtained an internal amino acid sequence and identified the 40 kDa protein as nucleolar protein B23. Western blotting experiments confirmed the internal amino acid sequencing results and showed both species (control, 5.4-5.8, irradiated, 5.2-5.5) to be recognized by an anti-B23 monoclonal antibody. Radiolabeling of control and irradiated samples with [32P]NAD or [32P]orthophosphoric acid revealed the acidic species of B23 to be both ADP-ribosylated and phosphorylated. Therefore, exposure of SQ-20B cells to radiation results in the increase in expression of an ADP-ribosylated and phosphorylated species of B23.

Enhanced expression of calreticulin in the nucleus of radioresistant squamous carcinoma cells in response to ionizing radiation.

Ionizing radiation has been shown to modulate gene and protein expression as well as cellular signal transduction pathways. However, the biochemical and molecular mechanisms that underlie the cellular response to radiation are not fully understood. The effects of ionizing radiation on the expression of nuclear proteins have now been investigated in radiorestistant human head and neck squamous carcinoma cells (SQ-20B cells) using the techniques of two-dimensional PAGE, silver staining, and computer-assisted quantitative analysis. Radiation (600 cGy) induced the expression of 10 proteins and suppressed the expression of 5 proteins in the nuclei of SQ-20B cells as detected 4 h after treatment. Electroelution and NH2-terminal amino acid sequence analysis revealed that one of the radiation-induced proteins was the Ca(2+)-binding protein calreticulin. The expression of calreticulin was increased approximately 6-fold in the nuclei of irradiated SQ-20B cells. Calreticulin and the other proteins whose expression was affected by radiation may contribute to the radioresistance phenotype of SQ-20B cells.

Co-regulated expression of dbl and poly(ADP-ribose) polymerase in Ewing's sarcoma cells and dbl-transformed NIH3T3 fibroblasts.

Poly(ADP-ribose) polymerase (PADPRP) is a ubiquitous enzyme constitutively expressed at low levels in the majority of eukaryotic cells, including most mammalian tumors and tumor-derived cell lines. Overexpression of PADPRP following the introduction of cDNA recombinant constructs into various cell types results in cell death. An exception to this effect are Ewing's sarcoma (ES) cells, which have been shown to contain elevated steady-state levels of PADPRP mRNA and high constitutive levels of protein and polymerase activity. In fact, this excess of PADPRP has been suggested to participate in the intrinsic radiosensitivity of Ewing's sarcomas, a highly malignant childhood bone tumor frequently curable with radiotherapy. It appears that ES cells might possess a hitherto unknown mechanism(s) by which PADPRP overexpression is controlled and made compatible with cell survival and proliferation. In order to investigate the contribution of other genetic alterations to PADPRP regulation in ES cells, we analysed the expression levels of PADPRP and of other genes, such as oncogenes and tumor suppressor genes, which may enhance the proliferative potential of ES cells. We have detected a positive correlation between the expression levels of the DNA-repair enzyme poly(ADP-ribose) polymerase and the dbl proto-oncogene in Ewing's sarcoma cells. The co-regulated expression of these genes has been established in NIH3T3 cells transformed by the human dbl oncogene or by overexpression of the dbl proto-oncogene. In both instances, the increase in dbl expression resulted in elevated levels of PADPRP mRNA and polymerase activity. The dbl oncogene was more efficient than the proto-oncogene in upregulating PADPRP expression. The inability of other oncogenes to upregulate PADPRP upon transformation of NIH3T3 cells demonstrated the specificity of the dbl in the process. Transfection of dbl-transformed NIH3T3 cells with retroviral PADPRP vectors resulted in the establishment of clones with PADPRP levels higher than those detectable in untransformed NIH3T3 cells transfected with the same retroviral constructs. These results suggest that dbl plays a role in the mechanism by which mammalian cells autoregulate their endogenous levels of PADPRP. Post-translational modification of the dbl or proto-dbl proteins by cytoplasmic PADPRP does not participate in the mechanism(s) underlying the observed PADPRP/dbl co-regulation.

Modulation of proliferation and tumorigenic potential of cervical-carcinoma cells by the expression of sense and antisense p53.

Previous studies from our laboratory demonstrated that overexpression of wild-type p53 suppresses the growth and alters the differentiation pattern of HPV-immortalized keratinocytes (Woodworth et al: Cell Growth Differ 4: 367-376, 1993). In the present report we describe the use of sense and antisense p53 retroviral vectors to modulate the expression levels of the p53 tumor suppressor gene in cervical carcinoma cells. Representative cell lines (C-4 I, ME-180 and C-33 A) were selected for this study on the basis of their various expression levels of either wild-type or mutated p53, and also of their content of integrated human papillomavirus sequences. Our results show that p53 overexpression from the sense construct decreased the growth rate and tumorigenic potential of the HPV-negative C-4 I cell line, whereas it had no effect on the HPV-positive ME-180 and C-33 A cells. However, examination of the individual colonies early after transfection with sense constructs by light microscopy showed that overexpression of wild-type p53 in ME-180 induced significant morphological alterations resulting in cellular senescence. Nevertheless, the p53 overexpressing, ME-180-derived cell line established after selection did not exhibit any major changes compared to the parental cell line. In the case of C-33 A, the presence of high levels of mutant p53 prevented the exogenous wild-type p53 from causing any significant modulatory action on their proliferation or neoplastic phenotype.

Differential expression of proteins in radioresistant and radiosensitive human squamous carcinoma cells.

BACKGROUND: Previous studies support a genetic basis for cellular radioresistance. The associated biochemical and molecular events, however, are not fully understood. PURPOSE: We investigated the differential protein pattern as a molecular determinant of resistance or sensitivity of head and neck squamous carcinoma cells to ionizing radiation. METHODS: Using two-dimensional polyacrylamide gel electrophoresis followed by computer-assisted quantitative analysis, we compared the protein profiles of three relatively radioresistant and three relatively radiosensitive head and neck squamous carcinoma cell lines (previously characterized by in vitro and clinical parameters as radioresistant or radiosensitive) to determine which proteins were consistently expressed or enhanced in the radioresistant compared with the radiosensitive phenotype. RESULTS: Our analysis indicated that 14 proteins were preferentially expressed in the radio-resistant cell lines SQ-20B, JSQ-3, and SCC-35, with one protein (molecular mass of 92 kd and pI of 5.5) distinctly expressed in the radioresistant cell lines. Four proteins were enhanced by greater than 10-fold, three were enhanced fivefold to 10-fold, and six were enhanced twofold to fivefold in the radioresistant cell lines. In addition, we observed a second set of 15 proteins preferentially expressed in the radio-sensitive cell lines SQ-9G, SQ-38, and SCC-9. A 40-kd protein (pI 7.1) was distinctly expressed in the radiosensitive cell lines. The remaining radiosensitive cell-specific proteins were enhanced by greater than 10-fold (two proteins), fivefold to 10-fold (two proteins), or twofold to fivefold (10 proteins) compared with their counterparts in the radioresistant cell lysates. CONCLUSION: These results provide evidence for differential protein expression associated with phenotypic expression of cellular radioresistance or radiosensitivity. IMPLICATIONS: This study will facilitate the characterization of these proteins correlated with the radiation response-specific phenotype.

Evidence that the central region of glycoprotein IIIa participates in integrin receptor function.

We have obtained evidence that the ligand-recognition region of the integrin beta-subunit, platelet glycoprotein IIIa (GPIIIa), is discontinuous. Receptor function can be localized to residues near the N-terminus and to the central region of the polypeptide chain. The epitope recognized by our monoclonal antibody, CS-1, which substantially inhibits fibrin(ogen) binding to ADP- and thrombin-stimulated platelets [Ramsamooj, Doellgast & Hantgan (1990) Thromb. Res. 58, 577-592], is contained within residues 349-422 of GPIIIa. This sequence is adjacent to a proteinase-resistant domain of GPIIIa which is linked by disulphide bond(s) to an N-terminal segment near to the putative Arg-Gly-Asp recognition site [D'Souza, Ginsberg, Burke, Lam & Plow (1988) Science 242, 91-93]. Limited trypsin digestion of purified platelet GPIIIa yielded a mixture of two-chain molecules comprised of an N-terminal fragment disulphide-bonded to one of four fragments, which began at residues 299, 303, 353 or 423. Tryptic cleavage of the 300-422 segment correlated with loss of immunoreactivity with anti-GPIIIa monoclonal antibody, CS-1. Chymotrypsin cleavage of GPIIIa resulted in an N-terminal 19 kDa fragment joined by at least one intrachain cystine residue to a 46 kDa polypeptide beginning at residue 349. Partial reduction with dithiothreitol released the larger chymotryptic fragment with its epitope for CS-1 intact. These results have enabled us to localize the epitope recognized by our inhibitory monoclonal antibody, CS-1, to residues 349-422 of GPIIIa. Our data are consistent with a structure in which both the N-terminal and central regions of GPIIIa, which may be in close proximity in the functional GPIIb-IIIa complex, participate in ligand binding.

Inhibition of fibrin(ogen) binding to stimulated platelets by a monoclonal antibody specific for a conformational determinant of GPIIIa.

We have purified the integrin GPIIb:IIIa from the membrane fraction of human blood platelets by lentil lectin affinity chromatography followed by gel filtration chromatography. With purified GPIIb:IIIa as an antigen, we have produced monoclonal antibody CS-1, which immunoblotting demonstrates to be specific for native GPIIIa; disulfide bond reduction of GPIIIa resulted in loss of immunoreactivity. Radiolabelled ligand binding studies revealed that CS-1 recognized approximately 55,000 sites per platelet and bound with a Kd in the nanomolar range, independent of the state of platelet activation. Binding of CS-1 or its Fab fragment to ADP- and thrombin-stimulated gel filtered platelets caused a 2-3 fold inhibition of binding the soluble ligands fibrinogen and fibrin protofibrils. CS-1 also inhibited aggregation of ADP- and thrombin-stimulated platelets by 2- and 4-fold, respectively. Since CS-1 inhibits fibrin(ogen) interactions with GPIIb:IIIa, we propose that the conformationally dependent epitope on GPIIIa recognized by CS-1 constitutes a region of the receptor which is involved in fibrin(ogen) binding.

Orientation and specificity of fibrin protofibril binding to ADP-stimulated platelets.

We have investigated the molecular basis of platelet:fibrin binding by studying interactions between platelets and protofibrils, soluble two-stranded polymers of fibrin, which are intermediates on the fibrin assembly pathway. The specificity of these interactions was examined with transmission electron microscopy (TEM), which clearly showed thin fibers with lengths to 150 nm attached to the cell surface of normal, stimulated platelets. Immunogold electron microscopy using rabbit anti-human fibrinogen as the first stage antibody verified the identity of the surface-bound molecules, and the immunogold distribution paralleled that observed with the fibrin/fibrinogen molecules alone. Contacts between the ends of the fibers and the platelets were frequently observed, but lateral contacts were also evident. Given the diameter at the point of fibrin contact (18.2 +/- 1.3 nm), it is possible that several glycoprotein receptors were involved in binding each protofibril. Morphometric analyses demonstrated that normal platelets stimulated by ADP in the absence of exogenous fibrin(ogen) or in the presence of fibrin protofibrils and antibodies directed against the GPIIb/IIIa complex lacked this molecular layer on the surface. Neither protofibrils nor fibrin fibers adhered to the surface of Glanzmann's thrombasthenic platelets, as demonstrated by TEM and microfluorimetry. Synthetic peptides of sequence RGDS and HHLGGAKQAGDV effectively blocked the binding of protofibrils to the surface of normal, stimulated platelets while synthetic GHRP had no effect. These results provide direct evidence for multiple points of attachment between fibrin protofibrils and the glycoprotein IIb/IIIa complexes present in a functional conformation on the surface of normal, stimulated platelets.

Structures of sialylated fucosyl polylactosaminoglycans isolated from chronic myelogenous leukemia cells.

Polylactosaminoglycans were isolated from human chronic myelogenous leukemia cells and their structures were elucidated. The lactosaminoglycan saccharides were isolated by hydrazinolysis and fractionated by QAE-Sephadex. The structures of fractionated oligosaccharides were analyzed by fast atom bombardment-mass spectrometry and methylation before and after treatment with specific exoglycosidases, such as alpha 2----3 specific neuraminidase. Based on these experiments, the structures of sialyl polylactosaminoglycans of chronic myelogenous leukemia cells were found to contain the following unique structure which is absent in normal mature granulocytes: (formula; see text) In addition to this, chronic myelogenous leukemia polylactosaminoglycans can be distinguished from normal granulocyte polylactosaminoglycans by the following characteristics. Leukemic polylactosaminoglycans are (a) shorter, (b) more highly sialylated and contain fully sialylated, tetrasialosyl polylactosaminoglycans, (c) are less fucosylated at C-3 of N-acetylglucosamine of polylactosaminyl side chains, and (d) contain a significant amount of sialyl Lex, NeuNAc alpha 2----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----3, structure. These results indicate that chronic myelogenous leukemia cells express unique polylactosaminoglycan structures which are distinct from normal mature granulocytes.