Structural requirements for peptidic antagonists of the corticotropin-releasing factor receptor (CRFR): Development of CRFR2β-selective antisauvagine-30

Different truncated and conformationally constrained analogs of corticotropin-releasing factor (CRF) were synthesized on the basis of the amino acid sequences of human/rat CRF (h/rCRF), ovine CRF (oCRF), rat urocortin (rUcn), or sauvagine (Svg) and tested for their ability to displace [125I-Tyr0]oCRF or [125I-Tyr0]Svg from membrane homogenates of human embryonic kidney (HEK) 293 cells stably transfected with cDNA coding for rat CRF receptor, type 1 (rCRFR1), or mouse CRF receptor, type 2β (mCRFR2β). Furthermore, the potency of CRF antagonists to inhibit oCRF- or Svg-stimulated cAMP production of transfected HEK 293 cells expressing either rCRFR1 (HEK-rCRFR1 cells) or mCRFR2β (HEK-mCRFR2β cells) was determined. In comparison with astressin, which exhibited a similar affinity to rCRFR1 (Kd = 5.7 ± 1.6 nM) and mCRFR2β (Kd = 4.0 ± 2.3 nM), [dPhe11,His12]Svg(11–40), [dLeu11]Svg(11–40), [dPhe11]Svg(11–40), and Svg(11–40) bound, respectively, with a 110-, 80-, 68-, and 54-fold higher affinity to mCRFR2β than to rCRFR1. The truncated analogs of rUcn displayed modest preference (2- to 7-fold) for binding to mCRFR2β. In agreement with the results of these binding experiments, [dPhe11,His12]Svg(11–40), named antisauvagine-30, was the most potent and selective ligand to suppress agonist-induced adenylate cyclase activity in HEK cells expressing mCRFR2β.

β subunits influence the biophysical and pharmacological differences between P- and Q-type calcium currents expressed in a mammalian cell line

Human epithelial kidney cells (HEK) were prepared to coexpress α1A, α2δ with different β calcium channel subunits and green fluorescence protein. To compare the calcium currents observed in these cells with the native neuronal currents, electrophysiological and pharmacological tools were used conjointly. Whole-cell current recordings of human epithelial kidney α1A-transfected cells showed small inactivating currents in 80 mM Ba2+ that were relatively insensitive to calcium blockers. Coexpression of α1A, βIb, and α2δ produced a robust inactivating current detected in 10 mM Ba2+, reversibly blockable with low concentration of ω-agatoxin IVA (ω-Aga IVA) or synthetic funnel-web spider toxin (sFTX). Barium currents were also supported by α1A, β2a, α2δ subunits, which demonstrated the slowest inactivation and were relatively insensitive to ω-Aga IVA and sFTX. Coexpression of β3 with the same combination as above produced inactivating currents also insensitive to low concentration of ω-Aga IVA and sFTX. These data indicate that the combination α1A, βIb, α2δ best resembles P-type channels given the rate of inactivation and the high sensitivity to ω-Aga IVA and sFTX. More importantly, the specificity of the channel blocker is highly influenced by the β subunit associated with the α1A subunit.

Ligand-independent oligomerization of cell-surface erythropoietin receptor is mediated by the transmembrane domain

Binding of erythropoietin (Epo) to the Epo receptor (EpoR) is crucial for production of mature red cells. Although it is well established that the Epo-bound EpoR is a dimer, it is not clear whether, in the absence of ligand, the intact EpoR is a monomer or oligomer. Using antibody-mediated immunofluorescence copatching (oligomerizing) of epitope-tagged receptors at the surface of live cells, we show herein that a major fraction of the full-length murine EpoR exists as preformed dimers/oligomers in BOSC cells, which are human embryo kidney 293T-derived cells. This observed oligomerization is specific because, under the same conditions, epitope-tagged EpoR did not oligomerize with several other tagged receptors (thrombopoietin receptor, transforming growth factor β receptor type II, or prolactin receptor). Strikingly, the EpoR transmembrane (TM) domain but not the extracellular or intracellular domains enabled the prolactin receptor to copatch with EpoR. Preformed EpoR oligomers are not constitutively active and Epo binding was required to induce signaling. In contrast to tyrosine kinase receptors (e.g., insulin receptor), which cannot signal when their TM domain is replaced by the strongly dimerizing TM domain of glycophorin A, the EpoR could tolerate the replacement of its TM domain with that of glycophorin A and retained signaling. We propose a model in which TM domain-induced dimerization maintains unliganded EpoR in an inactive state that can readily be switched to an active state by physiologic levels of Epo.

Synthesis and characterization of a photoactivatable analog of corticotropin-releasing factor for specific receptor labeling.

A novel photoactivatable analog of ovine corticotropin-releasing factor (ovine photoCRF) has been synthesized and characterized. A diazirine group, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl residue, was covalently bound to the amino terminus of ovine CRF (oCRF), which was N-terminally extended by a tyrosyl residue for radioactive labeling with 125I. Under mild conditions, photolysis yielded highly reactive carbenes, responsible for the formation of covalent bonds to the CRF receptor. Ovine photoCRF was shown to bind to the high-affinity site of the CRF receptor with a similar Kd value as oCRF. When radioactively iodinated ovine photoCRF (ovine 125I-photoCRF) was covalently linked to rat CRF receptor, type 1 (rCRFR1), permanently transfected into human embryonic kidney (HEK) 293 cells, a highly glycosylated 75-kDa protein was identified with SDS/PAGE. The specificity of ovine 125I-photoCRF was demonstrated by the finding that this analog could be displaced from the receptor by oCRF, but not other unrelated peptides such as vasoactive intestinal peptide. The observed size of the 75-kDa cross-link was in agreement with the molecular weight reported earlier for native CRFR1 from rat brain. Deglycosylation of the 75-kDa cross-link with peptide:N-glycosidase (PNGase) yielded a 46-kDa protein, in agreement with the molecular weight estimated from cDNA coding for rat CRFR1. The developed CRF analog, photoCRF, is expected to facilitate future biochemical and physiological analysis of CRF receptors and--by analogous strategies--of other peptide receptors.

Involvement of Asn-293 in stereospecific agonist recognition and in activation of the beta 2-adrenergic receptor.

To investigate the molecular mechanism for stereospecific binding of agonists to beta 2-adrenergic receptors we used receptor models to identify potential binding sites for the beta-OH-group of the ligand, which defines the chiral center. Ser-165, located in transmembrane helix IV, and Asn-293, situated in the upper half of transmembrane helix VI, were identified as potential binding sites. Mutation of Ser-165 to Ala did not change the binding of either isoproterenol isomer as revealed after transient expression in human embryonic kidney (HEK)-293 cells. In contrast, a receptor mutant in which Asn-293 was replaced by Leu showed substantial loss of stereospecific isoproterenol binding. Adenylyl cyclase stimulation by this mutant after stable expression in CHO cells confirmed the substantial loss of stereospecificity for isoproterenol. In a series of agonists the loss of affinity in the Leu-293 mutant receptor was strongly correlated with the intrinsic activity of the compounds. Full agonists showed a 10-30-fold affinity loss, whereas partial agonists had almost the same affinity for both receptors. Stereospecific recognition of antagonists was unaltered in the Leu-293 mutant receptor. These data indicate a relationship between stereospecificity and intrinsic activity of agonists and suggest that Asn-293 is important for both properties of the agonist-receptor interaction.

Radioimmunotherapy with a 64Cu-labeled monoclonal antibody: a comparison with 67Cu.

67Cu (t1/2 = 62 h) has demonstrated potential as a radionuclide for radioimmunotherapy, but limited availability severely restricts its widespread use. 64Cu (t1/2 = 12.8 h) has been shown to have comparable effectiveness in vitro and in vivo. The present study was undertaken to examine the therapeutic potential of 64Cu- and 67Cu-bromoacetamidobenzyl-1,4,8,11-tetraazacyclotetradeca ne-N, N',N",N"'-tetraacetic acid (BAT)-2-iminothiolane (2IT)-1A3 (1A3 is a mouse anti-human colorectal cancer mAb) for treatment of GW39 human colon carcinoma carried in hamster thighs. Hamsters were injected with 64Cu- or 67Cu-BAT-2IT-1A3 or Cu-labeled nonspecific IgG (MOPC) or saline. Hamsters were killed 6-7 months after therapy or when tumors were > or = 10 g. Of the hamsters with small tumors (mean weight 0.43 +/- 0.25 g), 87.5% were disease-free 7 months after treatment with 2 mCi (1 Ci = 37 GBq) of 64Cu-BAT-2IT-1A3 or 0.4 MCi of 67Cu-BAT-2IT-1A3. The mean tumor doses at these activities of 64Cu- and 67Cu-BAT-2IT-1A3 were 586 and 1269 rad (1 rad = 0.01 Gy), respectively. In contrast, 76% of hamsters treated with 2 mCi of 64Cu-BAT-2IT-MOPC or 0.4 mCi of 67Cu-BAT-2IT-MOPC had to be killed before 6 months because of tumor regrowth. When hamsters with larger tumors (mean weight 0.66 +/- 0.11 g) were treated with 64Cu- or 67Cu-BAT-2IT-1A3, survival was extended compared with controls, but only one animal remained tumor-free to 6 months. These results demonstrate that 64Cu- and 67Cu-BAT-2IT-1A3 given in a single administered dose can eradicate small tumors without significant host toxicity, but additional strategies to deliver higher tumor doses will be needed for larger tumors.

Association of the "major histocompatibility complex subregion" I-J determinant with bioactive glycosylation-inhibiting factor.

Murine suppressor T-cell hybridoma cells (231F1) secrete not only bioactive glycosylation-inhibiting factor (GIF) but also an inactive peptide comparable to bioactive GIF peptide in its molecular size and reactivity with anti-GIF; the amino acid sequence of the inactive peptide is identical to that of the bioactive homologue. The inactive GIF peptide in culture supernatant of both the 231F1 cells and a stable transfectant of human GIF cDNA in the murine suppressor T hybridoma selectively bound to Affi-Gel 10, whereas bioactive GIF peptides from the same sources failed to bind to the gel. The inactive cytosolic human GIF from the stable transfectant and Escherichia coli-derived recombinant human GIF also had affinity for Affi-Gel 10. Both the bioactive murine GIF peptide from the suppressor T hybridoma and bioactive recombinant human GIF from the stable transfectant bound to the anti-I-J monoclonal antibody H6 coupled to Affi-Gel. However, bioactive hGIF produced by a stable transfectant of human GIF cDNA in BMT10 cells failed to be retained in H6-coupled Affi-Gel. These results indicate that the I-J specificity is determined by the cell source of the GIF peptide and that the I-J determinant recognized by monoclonal antibody H6 does not represent a part of the primary amino acid sequence of GIF. It appears that the epitope is generated by a posttranslational modification of the peptide.

Radiometal labeling of recombinant proteins by a genetically engineered minimal chelation site: technetium-99m coordination by single-chain Fv antibody fusion proteins through a C-terminal cysteinyl peptide.

We describe a method to facilitate radioimaging with technetium-99m (99mTc) by genetic incorporation of a 99mTc chelation site in recombinant single-chain Fv (sFv) antibody proteins. This method relies on fusion of the sFv C terminus with a Gly4Cys peptide that specifically coordinates 99mTc. By using analogues of the 26-10 anti-digoxin sFv as our primary model, we find that addition of the chelate peptide, to form 26-10-1 sFv', does not alter the antigen-binding affinity of sFv. We have demonstrated nearly quantitative chelation of 0.5-50 mCi of 99mTc per mg of 26-10-1 sFv' (1 Ci = 37 GBq). These 99mTc-labeled sFv' complexes are highly stable to challenge with saline buffers, plasma, or diethylenetriaminepentaacetic acid. We find that the 99mTc-labeled 741F8-1 sFv', specific for the c-erbB-2 tumor-associated antigen, is effective in imaging human ovarian carcinoma in a scid mouse tumor xenograft model. This fusion chelate methodology should be applicable to diagnostic imaging with 99mTc and radioimmunotherapy with 186Re or 188Re, and its use could extend beyond the sFv' to other engineered antibodies, recombinant proteins, and synthetic peptides.

Molecular cloning and characterization of a conserved nuclear serine(threonine) protein kinase.

Human, Drosophila melanogaster, and Caenorhabditis elegans cDNA clones encoding homologues of a serine(threonine) protein kinase (EC 2.7.1.37) (designated Ndr protein kinase) have been isolated and sequenced. The human and Drosophila cDNAs predict polypeptides of 54 kDa and 52 kDa, respectively, which share approximately 80% amino acid similarity. Northern analysis of human tissues revealed a ubiquitously expressed 3.9-kb transcript. Recombinant GST-Ndr underwent intramolecular autophosphorylation on serine and threonine residues in vitro but failed to transphosphorylate several standard protein kinase substrates. Transfection of the human cDNA into COS-1 cells resulted in the appearance of an intense nuclear staining in cells analyzed by indirect immunofluorescence; deletion mutagenesis identified a short basic peptide, KRKAETWKRNRR, responsible for the nuclear accumulation of Ndr. Thus, Ndr is a conserved and widely expressed nuclear protein kinase. The closest known relative of this previously uncharacterized kinase is Dbf2, a budding yeast protein kinase required for the completion of nuclear division.

Wild-type p53 triggers a rapid senescence program in human tumor cells lacking functional p53

The p53 tumor suppressor gene has been shown to play an important role in determining cell fate. Overexpression of wild-type p53 in tumor cells has been shown to lead to growth arrest or apoptosis. Previous studies in fibroblasts have provided indirect evidence for a link between p53 and senescence. Here we show, using an inducible p53 expression system, that wild-type p53 overexpression in EJ bladder carcinoma cells, which have lost functional p53, triggers the rapid onset of G1 and G2/M growth arrest associated with p21 up-regulation and repression of mitotic cyclins (cyclin A and B) and cdc2. Growth arrest in response to p53 induction became irreversible within 48-72 h, with cells exhibiting morphological features as well as specific biochemical and ultrastructural markers of the senescent phenotype. These findings provide direct evidence that p53 overexpression can activate the rapid onset of senescence in tumor cells.

Taxol Suppresses Dynamics of Individual Microtubules in Living Human Tumor Cells

Microtubules are intrinsically dynamic polymers, and their dynamics play a crucial role in mitotic spindle assembly, the mitotic checkpoint, and chromosome movement. We hypothesized that, in living cells, suppression of microtubule dynamics is responsible for the ability of taxol to inhibit mitotic progression and cell proliferation. Using quantitative fluorescence video microscopy, we examined the effects of taxol (30–100 nM) on the dynamics of individual microtubules in two living human tumor cell lines: Caov-3 ovarian adenocarcinoma cells and A-498 kidney carcinoma cells. Taxol accumulated more in Caov-3 cells than in A-498 cells. At equivalent intracellular taxol concentrations, dynamic instability was inhibited similarly in the two cell lines. Microtubule shortening rates were inhibited in Caov-3 cells and in A-498 cells by 32 and 26%, growing rates were inhibited by 24 and 18%, and dynamicity was inhibited by 31 and 63%, respectively. All mitotic spindles were abnormal, and many interphase cells became multinucleate (Caov-3, 30%; A-498, 58%). Taxol blocked cell cycle progress at the metaphase/anaphase transition and inhibited cell proliferation. The results indicate that suppression of microtubule dynamics by taxol deleteriously affects the ability of cancer cells to properly assemble a mitotic spindle, pass the metaphase/anaphase checkpoint, and produce progeny.

Activation of p53 in cervical carcinoma cells by small molecules

In over 90% of cervical cancers and cancer-derived cell lines, the p53 tumor suppressor pathway is disrupted by human papillomavirus (HPV). The HPV E6 protein promotes the degradation of p53 and thus inhibits the stabilization and activation of p53 that would normally occur in response to HPV E7 oncogene expression. Restoration of p53 function in these cells by blocking this pathway should promote a selective therapeutic affect. Here we show that treatment with the small molecule nuclear export inhibitor, leptomycin B, and actinomycin D leads to the accumulation of transcriptionally active p53 in the nucleus of HeLa, CaSki, and SiHa cells. Northern blot analyses showed that both actinomycin D and leptomycin B reduced the amount of HPV E6-E7 mRNA whereas combined treatment with the drugs showed almost complete disappearance of the viral mRNA. The combined treatment activated p53-dependant transcription, and increases in both p21WAF1/CIP1 and Hdm2 mRNA were seen. The combined treatment resulted in apoptotic death in the cells, as evidenced by nuclear fragmentation and PARP-cleavage indicative of caspase 3 activity. These effects were greatly reduced by expressing a dominant negative p53 protein. The present study shows that small molecules can reactivate p53 in cervical carcinoma cells, and this reactivation is associated with an extensive biological response, including the induction of the apoptotic death of the cells.

Expression of the telomerase catalytic subunit, hTERT, induces resistance to transforming growth factor β growth inhibition in p16INK4A(−) human mammary epithelial cells

Failures to arrest growth in response to senescence or transforming growth factor β (TGF-β) are key derangements associated with carcinoma progression. We report that activation of telomerase activity may overcome both inhibitory pathways. Ectopic expression of the human telomerase catalytic subunit, hTERT, in cultured human mammary epithelial cells (HMEC) lacking both telomerase activity and p16INK4A resulted in gaining the ability to maintain indefinite growth in the absence and presence of TGF-β. The ability to maintain growth in TGF-β was independent of telomere length and required catalytically active telomerase capable of telomere maintenance in vivo. The capacity of ectopic hTERT to induce TGF-β resistance may explain our previously described gain of TGF-β resistance after reactivation of endogenous telomerase activity in rare carcinogen-treated HMEC. In those HMEC that overcame senescence, both telomerase activity and TGF-β resistance were acquired gradually during a process we have termed conversion. This effect of hTERT may model a key change occurring during in vivo human breast carcinogenesis.

Loss of oncogenic ras expression does not correlate with loss of tumorigenicity in human cells.

ras oncogenes are mutated in at variety of human tumors, which suggests that they play an important role in human carcinogenesis. To determine whether continued oncogenic ras expression is necessary to maintain the malignant phenotype, we studied the human fibrosarcoma cell line, HT1080, which contains one mutated and one wild-type N-ras allele. We isolated a variant of this cell line that no longer contained the mutated copy of the N-ras gene. Loss of mutant N-ras resulted in cells that displayed a less transformed phenotype characterized by a flat morphology, decreased growth rate, organized actin stress fibers, and loss of anchorage-independent growth. The transformed phenotype was restored following reintroduction of mutant N-ras. Although loss of the oncogenic N-ras drastically affected in vitro growth parameters, the variant remained tumorigenic in nude mice indicating that mutated N-ras expression is not necessary for maintenance of the tumorigenic phenotype. We confirmed this latter observation in colon carcinoma cell lines that have lost activated K-ras expression via targeted knockout of the mutant K-ras gene.

Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA.

We previously demonstrated that the putative oncogene AKT2 is amplified and overexpressed in some human ovarian carcinomas. We have now identified amplification of AKT2 in approximately 10% of pancreatic carcinomas (2 of 18 cell lines and 1 of 10 primary tumor specimens). The two cell lines with altered AKT2 (PANC1 and ASPC1) exhibited 30-fold and 50-fold amplification of AKT2, respectively, and highly elevated levels of AKT2 RNA and protein. PANC1 cells were transfected with antisense AKT2, and several clones were established after G418 selection. The expression of AKT2 protein in these clones was greatly decreased by the antisense RNA. Furthermore, tumorigenicity in nude mice was markedly reduced in PANC1 cells expressing antisense AKT2 RNA. To examine further whether overexpression of AKT2 plays a significant role in pancreatic tumorigenesis, PANC1 cells and ASPC1 cells, as well as pancreatic carcinoma cells that do not overexpress AKT2 (COLO 357), were transfected with antisense AKT2, and their growth and invasiveness were characterized by a rat tracheal xenotransplant assay. ASPC1 and PANC1 cells expressing antisense AKT2 RNA remained confined to the tracheal lumen, whereas the respective parental cells invaded the tracheal wall. In contrast, no difference was seen in the growth pattern between parental and antisense-treated COLO 357 cells. These data suggest that overexpression of AKT2 contributes to the malignant phenotype of a subset of human ductal pancreatic cancers.