Modulation of tumor eIF4E by antisense inhibition: A phase I/II translational clinical trial of ISIS 183750-an antisense oligonucleotide against eIF4E-in combination with irinotecan in solid tumors and irinotecan-refractory colorectal cancer.

The eukaryotic translation initiation factor 4E (eIF4E) is a potent oncogene that is found to be dysregulated in 30% of human cancer, including colorectal carcinogenesis (CRC). ISIS 183750 is a second-generation antisense oligonucleotide (ASO) designed to inhibit the production of the eIF4E protein. In preclinical studies we found that EIF4e ASOs reduced expression of EIF4e mRNA and inhibited proliferation of colorectal carcinoma cells. An additive antiproliferative effect was observed in combination with irinotecan. We then performed a clinical trial evaluating this combination in patients with refractory cancer. No dose-limiting toxicities were seen but based on pharmacokinetic data and tolerability the dose of irinotecan was reduced to 160 mg/m(2) biweekly. Efficacy was evaluated in 15 patients with irinotecan-refractory colorectal cancer. The median time of disease control was 22.1 weeks. After ISIS 183750 treatment, peripheral blood levels of eIF4E mRNA were decreased in 13 of 19 patients. Matched pre- and posttreatment tumor biopsies showed decreased eIF4E mRNA levels in five of nine patients. In tumor tissue, the intracellular and stromal presence of ISIS 183750 was detected by IHC in all biopsied patients. Although there were no objective responses stable disease was seen in seven of 15 (47%) patients who were progressing before study entry, six of whom were stable at the time of the week 16 CT scan. We were also able to confirm through mandatory pre- and posttherapy tumor biopsies penetration of the ASO into the site of metastasis.

Selumetinib with and without erlotinib in KRAS mutant and KRAS wild-type advanced nonsmall-cell lung cancer.

BACKGROUND: KRAS mutations in NSCLC are associated with a lack of response to epidermal growth factor receptor inhibitors. Selumetinib (AZD6244; ARRY-142886) is an oral selective MEK kinase inhibitor of the Ras/Raf/MEK/ERK pathway. PATIENTS AND METHODS: Advanced nonsmall-cell lung cancer (NSCLC) patients failing one to two prior regimens underwent KRAS profiling. KRAS wild-type patients were randomized to erlotinib (150 mg daily) or a combination of selumetinib (150 mg daily) with erlotinib (100 mg daily). KRAS mutant patients were randomized to selumetinib (75 mg b.i.d.) or the combination. The primary end points were progression-free survival (PFS) for the KRAS wild-type cohort and objective response rate (ORR) for the KRAS mutant cohort. Biomarker studies of ERK phosphorylation and immune subsets were carried out. RESULTS: From March 2010 to May 2013, 89 patients were screened; 41 KRAS mutant and 38 KRAS wild-type patients were enrolled. Median PFS in the KRAS wild-type arm was 2.4 months [95% confidence interval (CI) 1.3-3.7] for erlotinib alone and 2.1 months (95% CI 1.8-5.1) for the combination. The ORR in the KRAS mutant group was 0% (95% CI 0.0% to 33.6%) for selumetinib alone and 10% (95% CI 2.1% to 26.3%) for the combination. Combination therapy resulted in increased toxicities, requiring dose reductions (56%) and discontinuation (8%). Programmed cell death-1 expression on regulatory T cells (Tregs), Tim-3 on CD8+ T cells and Th17 levels were associated with PFS and overall survival in patients receiving selumetinib. CONCLUSIONS: This study failed to show improvement in ORR or PFS with combination therapy of selumetinib and erlotinib over monotherapy in KRAS mutant and KRAS wild-type advanced NSCLC. The association of immune subsets and immune checkpoint receptor expression with selumetinib may warrant further studies.

P2-purinergic receptor agonists inhibit the growth of androgen-independent prostate carcinoma cells.

To develop a new approach to the treatment of advanced, hormone-refractory prostate cancer, the signal transductions regulating the growth of human androgen-independent prostate carcinoma cell lines were studied. Agonist-stimulated Ca2+ mobilization, a critical regulatory event in other secretory cell types, was studied as a means of identifying previously undescribed plasma membrane receptors that may transduce a growth inhibitory signal. In all of the cell lines tested, P2-purinergic receptor agonists, including ATP and certain hydrolysis-resistant adenine nucleotides, induced a rapid, transient increase in cytoplasmic free Ca2+ that was detectable at 50 to 100 nM ATP, was maximal at 100 microM ATP, and was inhibited approximately 50% by chelation of extracellular Ca2+. Within 8 s after addition, ATP stimulated accumulation of the polyphosphatidylinositol products inositol (1, 4, 5) trisphosphate, inositol (1, 3, 4) trisphosphate, and inositol tetrakisphosphate. In addition to stimulating phosphatidylinositol turnover and Ca2+ mobilization, ATP and hydrolysis-resistant ATP analogues induced greater than 90% inhibition of the growth of all lines tested. These data demonstrate that human androgen-independent prostate carcinoma cells express functional P2-purinergic receptors linked to phospholipase C, and that agonists of this receptor are markedly growth inhibitory, suggesting a novel therapeutic approach to this common adult neoplasm.

In vitro enhancement of immunoglobulin gene expression in chronic lymphocytic leukemia.

B cell chronic lymphocytic leukemia (CLL) cells appear to be arrested in their differentiation so that little immunoglobulin is secreted in most cases. To determine their capacity for further differentiation we stimulated cells from a series of 10 cases of CLL with a phorbol ester and assayed for production of immunoglobulin protein, accumulation of immunoglobulin mRNA, and alterations in cell surface markers. We found that cells from all cases were induced to secret monoclonal immunoglobulin of the same heavy and light chain type as the surface membrane immunoglobulin type. Immunoglobulin secretion was preceded by a rapid increase in the levels of mRNA coding for IgM, predominantly the secretory form, mu s-mRNA, rather than the membrane form, mu m-mRNA. A similar selection of mu s- over mu m-mRNA is known to occur in plasma cells by a mechanism of differential processing of mRNA from a single mu-chain gene. Except for a decline in the expression of surface IgD, cell surface determinants remained unaffected both in terms of the percentage of positive cells and the relative number of sites per cell. In contrast to previous studies, these results indicate that CLL cells consistently retain the capacity to further differentiate toward plasma cells and secrete immunoglobulin. The immunoglobulin secretion is mediated, at least in part, by a developmentally regulated increment in mu s-mRNA.

Phorbol ester induces c-sis gene transcription in stem cell line K-562.

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced megakaryoblastic differentiation and c-sis expression in the human hematopoietic stem cell line K-562. This induction occurred at the transcriptional level, as determined by a nuclear runoff transcriptional assay, and was not a generalized effect of TPA, since the treatment of other hematopoietic cell lines and normal peripheral blood lymphocytes with TPA did not result in the appearance of c-sis mRNA.

Diltiazem inhibits transferrin receptor expression and causes G1 arrest in normal and neoplastic T cells.

Transferrin receptor expression is essential for the proliferation of both normal and malignant T cells. While transferrin receptor expression in normal T cells is tightly coupled to interleukin-2 receptor expression, transferrin receptor expression in malignant cells is usually constitutive and is released from this constraint. Temporally, the appearance of these membrane receptors is preceded by changes in the expression of the proto-oncogenes c-myc and c-myb. In addition, although an increase in the level of intracellular free calcium occurs early in the sequence of T-cell activation, the activation events dependent on this calcium flux have not been resolved. In the present study we report that diltiazem, an ion channel-blocking agent that inhibits calcium influx, arrested the growth in vitro of both normal and malignant human T cells in the G1 phase of the cell cycle. However, diltiazem did not inhibit the expression of c-myc or interleukin-2 receptor mRNA and protein in normal mitogen-activated T cells or the constitutive expression of c-myc and c-myb mRNA in malignant T cells (T acute lymphoblastic leukemia cells). In contrast, diltiazem prevented the induction of transferrin receptor (mRNA and protein) in normal T cells and caused a progressive loss of transferrin receptor (mRNA and protein) in malignant T cells. These data demonstrate that diltiazem can dissociate several growth-related processes normally occurring in G1 and thereby disrupt the biochemical cascade leading to cell proliferation.

Transcriptional inactivation of c-myc and the transferrin receptor in dibutyryl cyclic AMP-treated HL-60 cells.

Treatment of HL-60 cells with dibutyryl cyclic AMP induced rapid transcriptional inactivation of c-myc and the transferrin receptor. Transcriptional inactivation was followed by loss of c-myc and transferrin receptor mRNA and protein. Treated cells completed one round of proliferation, followed by growth arrest, G1 synchronization, and monocytic differentiation. These data suggest that cyclic AMP-mediated control of growth and differentiation may be achieved, at least in part, by transcriptional regulation of certain growth-associated proto-oncogenes and growth factor receptor genes.

Destabilization of Raf-1 by geldanamycin leads to disruption of the Raf-1-MEK-mitogen-activated protein kinase signalling pathway.

The serine/threonine kinase Raf-1 functions downstream of Rats in a signal transduction cascade which transmits mitogenic stimuli from the plasma membrane to the nucleus. Raf-1 integrates signals coming from extracellular factors and, in turn, activates its substrate, MEK kinase. MEK activates mitogen-activated protein kinase (MAPK), which phosphorylates other kinases as well as transcription factors. Raf-1 exists in a complex with HSP90 and other proteins. The benzoquinone ansamycin geldanamycin (GA) binds to HSP90 and disrupts the Raf-1-HSP90 multimolecular complex, leading to destabilization of Raf-1. In this study, we examined whether Raf-1 destabilization is sufficient to block the Raf-1-MEK-MAPK signalling pathway and whether GA specifically inactivates the Raf-1 component of this pathway. Using the model system of NIH 3T3 cells stimulated with phorbol 12-myristate 13-acetate (PMA), we show that GA does not affect the ability of protein kinase C alpha to be activated by phorbol esters, but it does block activation of MEK and MAPK. Further, GA does not decrease the activity of constitutively active MEK in transiently transfected cells. Finally, disruption of the Raf-1-MEK-MAPK signalling pathway by GA prevents both the PMA-induced proliferative response and PMA-induced activation of a MAPK-sensitive nuclear transcription factor. Thus, we demonstrate that interaction between HSP90 and Raf-1 is a sine qua non for Raf stability and function as a signal transducer and that the effects observed cannot be attributed to a general impairment of protein kinase function.

Various methods of analysis of mdr-1/P-glycoprotein in human colon cancer cell lines.

BACKGROUND: Multidrug resistance (MDR) mediated by high levels of mdr-1 (also known as PGY1)/P-glycoprotein (Pgp) has been studied in tissue culture systems; however, most tumor samples which express mdr-1/Pgp have much lower levels. PURPOSE: We wanted to determine if levels seen clinically could be detected by commonly used methods and to determine if these levels conferred MDR reversible by Pgp antagonists. METHODS: We studied multi-drug-resistant cell lines and sublines with levels of mdr-1/Pgp expression comparable to those seen clinically. We evaluated the expression of mdr-1 RNA by Northern blot analysis, slot blot analysis, polymerase chain reaction (PCR) analysis, and in situ hybridization. We evaluated protein expression by immunofluorescence, immunohistochemistry, fluorescence-activated cell sorting, and immunoblotting analyses. Drug resistance and reversibility were determined by cell growth during continuous drug exposure. RESULTS: In most cases, the low level of mdr-1/Pgp present in these cell lines could be detected by each method, but the assays were at the limit of sensitivity for all methods except the PCR method. These low levels of mdr-1/Pgp are capable of conferring MDR, which can be antagonized by verapamil. CONCLUSIONS: Levels of mdr-1/Pgp similar to those found in clinical samples can be detected by each of these methods, but the PCR method was the most sensitive and most reliably quantitative. IMPLICATIONS: In vitro sensitization by the addition of verapamil in cell lines with these low levels of mdr-1/Pgp suggests that clinically detected levels may confer drug resistance in vivo.

Taxol-induced apoptosis and phosphorylation of Bcl-2 protein involves c-Raf-1 and represents a novel c-Raf-1 signal transduction pathway.

c-Raf-1 (Raf-1) is a central component of signal transduction pathways stimulated by various growth factors, protein kinase C, and other protein kinases. Raf-1 activation is thought to be initiated at the plasma membrane after its recruitment by Ras. Raf-1 activation is associated primarily with proliferation and cell survival, but it has also been implicated in apoptosis. Raf-1 has also been shown to form complexes with both R-Ras and Bcl-2, raising the possibility that this component of cellular Raf-1 plays a role in apoptosis. Recently, taxol was reported to induce Bcl-2 phosphorylation and inactivation. We have previously demonstrated Raf-1 activation following taxol in MCF7 cells. We now present evidence that taxol fails to stimulate either apoptosis or phosphorylation of Bel-2 in the absence of Raf-1. Moreover, Raf-1 activation by taxol coincided with Bel-2 phosphorylation, showing similar dose and time dependence. Thus, our data support a role for a distinct subcellular component of Raf-1, which is taxol but not phorbol myristate acetate sensitive, in mediating an apoptotic pathway involving Bc1-2.

Immunological quantitation of thymidylate synthase using the monoclonal antibody TS 106 in 5-fluorouracil-sensitive and -resistant human cancer cell lines.

Thymidylate synthase (TS) (EC 2.1.1.45) is an important cellular target for the fluoropyrimidine cytotoxic drugs that are widely used in the treatment of solid tumors. Using the TS 106 monoclonal antibody to human TS, we have compared the immunological quantitation of TS by Western immunoblot and immunofluorescent techniques to the conventional biochemical 5-fluorodeoxyuridine monophosphate binding assay in a panel of 5-fluorouracil (5-FU)-sensitive and -resistant human cancer cell lines. Densitometric quantitation of TS 106-labeled Western immunoblot analysis of cell lysates from two 5-FU-resistant colon carcinoma cell lines, NCI H630R1 and NCI H630R10, revealed 12.8- and 16-fold increases in TS, respectively, compared to the parent 5-FU-sensitive NCI H630 colon cell line. By biochemical analysis, the TS level was 15- and 23-fold higher, respectively, in these resistant cell lines. Similarly, immunoblot analysis of cell lysates from two 5-FU-resistant breast cancer cell lines, MCF-Ad5 and MCF-Ad10, detected a 2.3- and 6.3-fold increase in TS, respectively, compared to the parent MCF-7 cell line. By biochemical assay the TS activity was 1.8- and 7.0-fold higher in these resistant breast cell lines. Western immunoblotting analysis revealed a 35-fold range of TS protein concentrations among the 10 cell lines examined, compared to a 38-fold range demonstrated by the biochemical assay. Direct comparison of Western blotting and the biochemical assay revealed a highly significant correlation (r2 = 0.93) between the two assays. Moreover, using the monoclonal antibody TS 106, the Western blotting technique was capable of detecting TS protein levels as low as 0.3 fmol in cellular lysates. Quantitation of TS in intact cells by immunofluorescent TS labeling and flow cytometric analysis was performed using three of the cell lines, NCI H630, NCI H630R10, and MCF-Ad10. This revealed a 26-fold increase in TS in the 5-FU-resistant NCI H630R10 line compared to the parent NCI H630 line and a 3.5-fold increase in TS compared to the 5-FU-resistant MCF-Ad10 breast cell line. The 5-FU-resistant MCF-Ad10 breast cell line, in turn, displayed a 7.7-fold increase in TS, compared to the 5-FU-sensitive NCI H630 cell lines. TS immunofluorescent analysis was capable of measuring TS within individual cells. The development of these immunological assays using an anti-TS monoclonal antibody will facilitate the quantitation of TS in cell lines and tissue samples.

Effect of guanine and adenine nucleotides on bombesin-stimulated phospholipase C activity in membranes from Swiss 3T3 and small cell lung carcinoma cells.

In [3H]inositol-labeled membranes prepared from Swiss mouse 3T3 and human small cell lung carcinoma cells, [Tyr4]-bombesin stimulated production of water-soluble inositol phosphates. The reaction was stimulated by guanosine 5'-O-[3-thiotriphosphate] and was specifically inhibited by both [Leu13-psi-CH2NHLeu14]-bombesin and the antibombesin antibody 2A11. [Tyr4]-bombesin-induced activation of phospholipase C is most apparent in Ca2(+)-depleted conditions (less than 1 microM[Ca2+]free). The kinetics of activation by ligand also demonstrate that [Tyr4]-bombesin-dependent phospholipase C activation is most apparent at [Mg2+]free of approximately 0.2 microM. At millimolar concentrations of [Mg2+]free, there is considerably less dependence on [Tyr4]-bombesin for activation of phospholipase C. ATP is not necessary for initial activation of phospholipase C, and beta, gamma-imidoadenosine-5'-triphosphate does not inhibit the reaction. These results demonstrate that in these cell types [Tyr4]-bombesin activates phospholipase C in conjunction with guanine nucleotides. Phospholipase C-coupled guanine nucleotide regulatory proteins would be appropriately considered as novel targets for the development of therapeutic strategies in small cell lung carcinoma.

Taxol induction of p21WAF1 and p53 requires c-raf-1.

Taxol stabilizes microtubules, prevents tubulin depolymerization, and promotes tubulin bundling and is one of the most effective drugs for the treatment of metastatic breast and ovarian cancer. Although its interaction with tubulin has been well characterized, the mechanism by which taxol induces growth arrest and cytotoxicity is not well understood. Herein, we show that taxol induced dose- and time-dependent accumulation of the cyclin inhibitor p21WAF1 in both p53 wild-type and p53-null cells, although the degree of induction was greater in cells expressing wild-type p53. In MCF7 cells, wild-type p53 protein was also induced after taxol treatment, and this induction was mediated primarily by increased protein stability. Taxol induced both p21WAF1 and wild-type p53 optimally in MCF7 cells after 20-24-h exposure with an EC50(3) of 5 nM. In p53-null PC3M cells, p21WAF1 was similarly induced after 24-h exposure to taxol. Coincident with these biochemical effects, taxol altered the electrophoretic mobility of c-raf-1 and stimulated mitogen activated protein kinase. Previous depletion of c-raf-1 inhibited both the p21WAF1- and p53-inducing properties of taxol, as well as the activation of MAP kinase. These data suggest that induction of p21WAF1 by taxol requires c-raf-1 activity, but that it is not strictly dependent on wild-type p53. Furthermore, the ability of taxol to both induce wild-type p53 in MCF7 cells and activate MAP kinase is also dependent on c-raf-1 expression.

Drug-induced apoptosis is not necessarily dependent on macromolecular synthesis or proliferation in the p53-negative human prostate cancer cell line PC-3.

The propensity of a cell to undergo apoptosis has been proposed to be a determinant for chemotherapy sensitivity that is not directly dependent on specific drug-target interactions. Androgen-independent prostate cancer is typically refractory to cytotoxic drugs, and we tested whether this is due to a loss of the ability to undergo apoptosis. Exposure of the hormone-insensitive and p53-negative human prostate carcinoma cell line PC-3 to 22 microM cisplatin, 1 microM camptothecin, 10 microM tenoposide, 135 nM vincristine, or 10 microM lovastatin for 72 h caused cell death, internucleosomal DNA fragmentation, and morphological changes typical for apoptosis. One microM cycloheximide prevented anticancer drug-induced apoptosis, whereas high concentration (1 mM) of cycloheximide alone induced apoptosis, indicating that protein synthesis was not needed for these cells to undergo apoptosis. Since cycloheximide affected DNA synthesis and proliferation of PC-3 cells, we tested whether the DNA polymerase inhibitor aphidicolin could also suppress drug-induced apoptosis. In contrast to cycloheximide, aphidicolin inhibited only vincristine-induced apoptosis. Cycloheximide prevented drug-induced changes in cell cycle distribution except for vincristine, while aphidicolin led to an accumulation of cells at the G1-S border independent of the drug used. These data indicate that macromolecular synthesis, active cell cycling, and p53 expression are not required for apoptosis to proceed in this system.

MS-275, a histone deacetylase inhibitor, selectively induces transforming growth factor beta type II receptor expression in human breast cancer cells.

Transcriptional repression of the transforming growth factor (TGF)-1P type II receptor (TPRII) gene appears to be a major mechanism to inactivate TGF-beta responsiveness in many human cancers. Because histone acetylation/deacetylation plays a role in transcriptional regulation, we have examined the effect of MS-275, a synthetic inhibitor of histone deacetylase, in human breast cancer cell lines. MS-275 showed antiproliferative activity against all human breast cancer cell lines examined and induced TbetaRII mRNA, but not TGF-beta type I receptor mRNA. MS-275 caused an accumulation of acetylated histones H3 and H4 in total cellular chromatin. An increase in the accumulation of acetylated histones H3 and H4 was detected in the TbetaRII promoter after treatment with MS-275. However, the level of histone acetylation did not change in chromatin associated with the TGF-beta type I receptor gene. MS-275 treatment enhanced TGF-beta1-induced plasminogen activator inhibitor 1 expression. Thus, antitumor activity of MS-275 may be mediated in part through the induction of TbetaRII expression and consequent potentiation of TGF-beta signaling.

Geldanamycin abrogates ErbB2 association with proteasome-resistant beta-catenin in melanoma cells, increases beta-catenin-E-cadherin association, and decreases beta-catenin-sensitive transcription.

Beta-catenin undergoes both serine and tyrosine phosphorylation. Serine phosphorylation in the amino terminus targets beta-catenin for proteasome degradation, whereas tyrosine phosphorylation in the COOH terminus influences interaction with E-cadherin. We examined the tyrosine phosphorylation status of beta-catenin in melanoma cells expressing proteasome-resistant beta-catenin, as well as the effects that perturbation of beta-catenin tyrosine phosphorylation had on its association with E-cadherin and on its transcriptional activity. Beta-catenin is tyrosine phosphorylated in three melanoma cell lines and associates with both the ErbB2 receptor tyrosine kinase and the LAR receptor tyrosine phosphatase. Geldanamycin, a drug which destabilizes ErbB2, caused rapid cellular depletion of the kinase and loss of its association with beta-catenin without perturbing either LAR or beta-catenin levels or LAR/beta-catenin association. Geldanamycin also stimulated tyrosine dephosphorylation of beta-catenin and increased beta-catenin/E-cadherin association, resulting in substantially decreased cell motility. Geldanamycin also decreased the nuclear beta-catenin level and inhibited beta-catenin-driven transcription, as assessed using two different beta-catenin-sensitive reporters and the endogenous cyclin D1 gene. These findings were confirmed by transient transfection of two beta-catenin point mutants, Tyr-654Phe and Tyr-654Glu, which, respectively, mimic the dephosphorylated and phosphorylated states of Tyr-654, a tyrosine residue contained within the beta-catenin-ErbB2-binding domain. These data demonstrate that the functional activity of proteasome-resistant beta-catenin is regulated further by geldanamycin-sensitive tyrosine phosphorylation in melanoma cells.

Metastasis-associated differences in gene expression in a murine model of osteosarcoma.

Despite advances in the management of osteosarcoma (OSA) and other solid tumors, the development of metastasis continues to be the most significant problem and cause of death for cancer patients. To define genetic determinants of pulmonary metastasis, we have applied cDNA microarrays to a recently described murine model of OSA that is characterized by orthotopic tumor growth, a period of minimal residual disease, spontaneous pulmonary metastasis, and cell line variants that differ in metastatic potential. Microarray analysis defined 53 genes (of 3166 unique cDNAs) that were differentially expressed between the primary tumors of the more aggressive (K7M2) and less aggressive (K12) OSA models. By review of the literature, these differentially expressed genes were assigned to six nonmutually exclusive metastasis-associated categories (proliferation and apoptosis, motility and cytoskeleton, invasion, immune surveillance, adherence, and angiogenesis). Functional studies to evaluate K7M2 and K12 for differences in each of these metastasis-associated processes revealed enhanced motility, adherence, and angiogenesis in the more aggressive K7M2 model. For this reason, 10 of the 53 differentially expressed genes that were assigned to the motility and cytoskeleton, adherence, and angiogenesis categories were considered as most likely to define differences in the metastatic behavior of the two models. Ezrin, a gene not described previously in OSA, with functions in motility, invasion, and adherence, was 3-fold overexpressed in K7M2 compared with K12 by microarray. Differential expression for RNA was confirmed by Northern analysis and for protein by immunostaining. Alterations in ezrin protein levels and concomitant cytoskeletal changes in our model confirmed predictions from the arrays. The potential relevance of ezrin in OSA was suggested by its expression in five of five human OSA cell lines. This work represents a rationale approach to the evaluation of microarray data and will be useful to identify genes that may be causally associated with metastasis.

Characteristics of cell lines established from human gastric carcinoma.

We report the establishment and characterization of four continuous cell lines derived from human primary and metastatic gastric carcinomas, and we compare their properties with a panel of colorectal carcinoma cell lines previously established and reported by us. Our success rate in culturing gastric carcinomas was relatively low, especially from primary tumors, compared to colorectal carcinoma. These observations may reflect the relatively modest number of gastric carcinoma cell lines established (mainly from Japan), compared to the abundance of colorectal carcinoma lines established worldwide. All four gastric lines expressed the surface glycoproteins carcinoembryonic antigen and TAG-72 and three lines expressed CA 19-9. Two of the lines expressed aromatic amino acid decarboxylase but lacked other markers for neuroendocrine differentiation. All four lines were positive for vasoactive intestinal peptide receptors but lacked gastrin receptors. In addition, two lines expressed receptors for muscarinic/cholinergic receptors but not beta-adrenergic receptors. Cytogenetic evidence for gene amplification was present in the cell lines. All four lines contained varying numbers of double-minute chromosomes. One line, SNU-16, was amplified for the c-myc proto-oncogene and contained four homogeneously staining regions. While c-myc and c-erb-B-2 RNA were expressed by all lines, there was no evidence of amplification or overexpression of several other proto-oncogenes and growth factors. The multiple properties we have described in our gastric carcinoma cell lines are remarkably similar to those found in the panel of colorectal carcinoma cell lines. These properties include morphology, growth characteristics, expression of surface glycoproteins, partial expression of neuroendocrine cell markers, frequent chromosomal evidence of gene amplification, and occasional amplification of the c-myc proto-oncogene. Our four well characterized cell lines should provide useful additions to the modest number currently available for in vitro studies of gastric carcinoma.

In vivo degradation of N-myc in neuroblastoma cells is mediated by the 26S proteasome.

N-myc is a short-lived transcription factor, frequently amplified in human neuroblastomas. The ubiquitin-proteasome system is involved in the degradation of many short-lived cellular proteins and previous studies have shown that ubiquitin-dependent proteolysis is implicated in the turn-over of N-myc in vitro. However, calpain has also been implicated in N-myc degradation in vitro. Here we report that, in vivo, N-myc is a sensitive substrate for the 26S proteasome in N-myc amplified neuroblastoma cells. We observed that inhibition of the 26S proteasome with two inhibitors, ALLnL and lactacystin, led to an elevation of the N-myc protein steady-state and increased N-myc protein polyubiquitination, as revealed by ubiquitin Western blotting. Pulse-chase experiments have shown that the increased N-myc levels resulted from stabilization of the protein. In contrast treatment with several calpain and cathepsin inhibitors failed to block N-myc degradation in vivo. Furthermore, fluorescence microscopy of ALLnL-treated cells localized N-myc exclusively to the nuclear compartment, suggesting the absence of a requirement for transport to the cytoplasm prior to degradation.

Regulation of BRCA1 by protein degradation.

BRCA1, a tumor suppressor protein implicated in hereditary forms of breast and ovarian cancer, is transcriptionally regulated in a proliferation-dependent manner. In this study, we demonstrate a substantial role for proteolysis in regulating the BRCA1 steady-state protein level in several cell lines. N-acetyl-leu-leu-norleucinal (ALLN), an inhibitor of the proteasome, calpain, and cathepsins, caused BRCA1 protein to accumulate in the nucleus of several human breast, prostate, and melanoma cell lines which express low or undetectable basal levels of BRCA1 protein, but not in cells with high basal expression of BRCA1. Protease inhibition did not increase BRCA1 synthesis, nor change its mRNA level, but it dramatically prolonged the protein's half-life. In contrast to ALLN, lactacystin and PS341, two specific proteasome inhibitors, as well as calpastatin peptide and PD150606, two selective calpain inhibitors, had no effect on BRCA1 stability, whereas ALLM, an effective calpain and cathepsin inhibitor but weak proteasome inhibitor, did stimulate accumulation of BRCA1. Moreover, three inhibitors of acidic cysteine proteases, chloroquine, ammonium chloride and bafilomycin, were as effective as ALLN. These results demonstrate that degradation by a cathepsin-like protease in fine balance with BRCA1 transcription is responsible for maintaining the low steady-state level of BRCA1 protein seen in many cancer cells.