Mammaglobin B (SCGB2A1) is a novel tumour antigen highly differentially expressed in all major histological types of ovarian cancer: implications for ovarian cancer immunotherapy.

BACKGROUND: We studied the genetic fingerprints of ovarian cancer and validated the potential of Mammaglobin b (SCGB2A1), one of the top differentially expressed genes found in our analysis, as a novel ovarian tumour rejection antigen. METHODS: We profiled 70 ovarian carcinomas including 24 serous (OSPC), 15 clear-cell (CC), 24 endometrioid (EAC) and 7 poorly differentiated tumours, and 14 normal human ovarian surface epithelial (HOSE) control cell lines using the Human HG-U133 Plus 2.0 chip (Affymetrix). Quantitative real-time PCR and immunohistochemistry staining techniques were used to validate microarray data at RNA and protein levels for SCGB2A1. Full-length human-recombinant SCGB2A1 was used to pulse monocyte-derived dendritic cells (DCs) to stimulate autologous SCGB2A1-specific cytotoxic T-lymphocyte (CTL) responses against chemo-naive and chemo-resistant autologous ovarian tumours. RESULTS: Gene expression profiling identified SCGB2A1 as a top differentially expressed gene in all histological ovarian cancer types tested. The CD8+ CTL populations generated against SCGB2A1 were able to consistently induce lysis of autologous primary (chemo-naive) and metastatic/recurrent (chemo-resistant) target tumour cells expressing SCGB2A1, whereas autologous HLA-identical noncancerous cells were not lysed. Cytotoxicity against autologous tumour cells was significantly inhibited by anti-HLA-class I (W6/32) monoclonal antibody. Intracellular cytokine expression measured by flow cytometry showed a striking type 1 cytokine profile (i.e., high IFN-γ secretion) in SCGB2A1-specific CTLs. CONCLUSION: SCGB2A1 is a top differentially expressed gene in all major histological types of ovarian cancers and may represent a novel and attractive target for the immunotherapy of patients harbouring recurrent disease resistant to chemotherapy.

Interaction of 125I-labeled botulinum neurotoxins with nerve terminals. II. Autoradiographic evidence for its uptake into motor nerves by acceptor-mediated endocytosis.

Using pharmacological (Simpson, L.L., 1980, J. Pharmacol. Exp. Ther. 212:16-21) and autoradiographic techniques (Black, J.D., and J.O. Dolly, 1986, J. Cell Biol., 103:521-534), it has been shown that botulinum neurotoxin (BoNT) is translocated across the motor nerve terminal membrane to reach a postulated intraterminal target. In the present study, the nature of this uptake process was investigated using electron microscopic autoradiography. It was found that internalization is acceptor-mediated and that binding to specific cell surface acceptors involves the heavier chain of the toxin. In addition, uptake was shown to be energy and temperature-dependent and to be accelerated by nerve stimulation, a treatment which also shortens the time course of the toxin-induced neuroparalysis. These results, together with the observation that silver grains were often associated with endocytic structures within the nerve terminal, suggested that acceptor-mediated endocytosis is responsible for toxin uptake. This proposal is supported further by the fact that lysosomotropic agents, which are known to interfere with the endocytic pathway, retard the onset of BoNT-induced neuroparalysis and also affect the distribution of silver grains at nerve terminals treated with 125I-BoNT. Possible recycling of BoNT acceptors (an important aspect of acceptor-mediated endocytosis of toxins) at motor nerve terminals was indicated by comparing the extent of labeling in the presence and absence of metabolic inhibitors. On the basis of these collective results, it is concluded that BoNT is internalized by acceptor-mediated endocytosis and, hence, the data support the proposal that this toxin inhibits release of acetylcholine by interaction with an intracellular target.

Interaction of 125I-labeled botulinum neurotoxins with nerve terminals. I. Ultrastructural autoradiographic localization and quantitation of distinct membrane acceptors for types A and B on motor nerves.

The labeling patterns produced by radioiodinated botulinum neurotoxin (125I-BoNT) types A and B at the vertebrate neuromuscular junction were investigated using electron microscopic autoradiography. The data obtained allow the following conclusions to be made. 125I-BoNT type A, applied in vivo or in vitro to mouse diaphragm or frog cutaneous pectoris muscle, interacts saturably with the motor nerve terminal only; silver grains occur on the plasma membrane, within the synaptic bouton, and in the axoplasm of the nerve trunk, suggesting internalization and retrograde intra-axonal transport of toxin or fragments thereof. 125I-BoNT type B, applied in vitro to the murine neuromuscular junction, interacts likewise with the motor nerve terminal except that a lower proportion of internalized radioactivity is seen. This result is reconcilable with the similar, but not identical, pharmacological action of these toxin types. The saturability of labeling in each case suggested the involvement of acceptors; on preventing the internalization step with metabolic inhibitors, their precise location became apparent. They were found on all unmyelinated areas of the nerve terminal membrane, including the preterminal axon and the synaptic bouton. Although 125I-BoNT type A interacts specifically with developing terminals of newborn rats, the unmyelinated plasma membrane of the nerve trunk is not labeled, indicating that the acceptors are unique components restricted to the nerve terminal area. BoNT types A and B have distinct acceptors on the terminal membrane. Having optimized the conditions for saturation of these binding sites and calibrated the autoradiographic procedure, we found the densities of the acceptors for types A and B to be approximately 150 and 630/micron 2 of membrane, respectively. It is proposed that these membrane acceptors target BoNT to the nerve terminal and mediate its delivery to an intracellular site, thus contributing to the toxin's selective inhibitory action on neurotransmitter release.

Activation of protein kinase C isozymes is associated with post-mitotic events in intestinal epithelial cells in situ.

The mechanisms underlying control of cell growth and differentiation in epithelial tissues are poorly understood. Protein kinase C (PKC) isozymes, members of a large family of serine/threonine kinases of fundamental importance in signal transduction, have been increasingly implicated in the regulation of cell growth, differentiation, and function. Using the rat intestinal epithelium as a model system, we have examined PKC-specific activity as well as individual PKC isozyme expression and distribution (i.e., activation status) in epithelial cells in situ. Increased PKC activity was detected in differentiating and functional cells relative to immature proliferating crypt cells. Immunofluorescence and Western blot analysis using a panel of isozyme-specific antibodies revealed that PKC alpha, beta II, delta, epsilon, and zeta are expressed in rat intestinal epithelial cells and exhibit distinct subcellular distribution patterns along the crypt-villus unit. The combined morphological and biochemical approach used permitted analysis of the activation status of specific PKC isozymes at the individual cell level. These studies showed that marked changes in membrane association and level of expression for PKC alpha, beta II, delta, and zeta occur as cells cease division in the mid-crypt region and begin differentiation. Additional changes in PKC activation status are observed with acquisition of mature function on the villus. These studies clearly demonstrate naturally occurring alterations in PKC isozyme activation status at the individual cell level within the context of a developing tissue. Direct activation of PKC in an immature intestinal crypt cell line was shown to result in growth inhibition and coincident translocation of PKC alpha from the cytosolic to the particulate subcellular fraction, paralleling observations made in situ and providing further support for a role of intestinal PKC isozymes in post-mitotic events. PKC isozymes were also found to be tightly associated with cytoskeletal elements, suggesting participation in control of the structural organization of the enterocyte. Taken together, the results presented strongly suggest an involvement of PKC isoforms in cellular processes related to growth cessation, differentiation, and function of intestinal epithelial cells in situ.

Heterogeneity in lymphocyte spectrin distribution: ultrastructural identification of a new spectrin-rich cytoplasmic structure.

Spectrin-like proteins are found in a wide variety of non-erythroid cells where they generally occur in the cell cortex near the plasma membrane. To determine the intracellular distribution of alpha-spectrin (alpha-fodrin) in lymphocytes, we have developed an immunoperoxidase method to localize this protein at the ultrastructural level. Of considerable interest, particularly with regard to our efforts to determine the function of spectrin in this cell type, was the finding that its subcellular localization and its relationship with the plasma membrane can vary dramatically. Based on its position in the cell, alpha-spectrin can occur in two forms in lymphocytes: one that associates closely with the plasma membrane and another that occurs at some distance from the cell periphery, either as a single large aggregate or as several smaller ones. The single large aggregate of spectrin is a stable feature in a number of lymphocyte cell lines and hybrids which were used to examine its ultrastructural characteristics. A previously undescribed cellular structure, consisting of a meshwork of spectrin filaments and membranous vesicles, was identified in these cells. This structure could be induced to dissipate in response to membrane perturbants (e.g., hyperthermia and phorbol esters, known effectors of lymphocyte function and differentiation) and the patterns resulting from the redistribution of spectrin were a reflection of those observed routinely in lymphocytes in situ. The correlation between naturally occurring spectrin localization patterns and those seen after membrane perturbation suggested the possibility that spectrin distribution is indicative of particular maturation stages or functional states in lymphocytes. The implications of these findings with regard to the role of spectrin in lymphocyte function are discussed.

Dynamic properties of ankyrin in T lymphocytes: colocalization with spectrin and protein kinase C beta.

Ankyrin is a well characterized membrane skeletal protein which has been implicated in the anchorage of specific integral membrane proteins to the spectrin-based membrane skeleton in a number of systems. In this study, the organization of ankyrin was examined in lymphocytes in relation to T cell function. Light and electron microscope immunolocalization studies revealed extensive heterogeneity in the subcellular distribution of ankyrin in murine tissue-derived lymphocytes. While ankyrin can be localized at the lymphocyte plasma membrane, it can also be accumulated at some distance from the cell periphery, in small patches or in a single discrete, nonmembrane-bound structure. Double immunofluorescence studies demonstrated that ankyrin colocalizes with spectrin and with the signal transducing molecule protein kinase C beta (PKC beta) in tissue-derived lymphocytes, suggesting a functional association between these molecules in the lymphocyte cytoplasm. In addition, T lymphocyte activation-related signals and phorbol ester treatment, both of which lead to PKC activation, cause a rapid translocation of ankyrin, together with spectrin and PKC beta, to a single Triton X-100-insoluble aggregate in the cytoplasm. This finding suggests a mechanism for the reported appearance of PKC in the particulate fraction of cells after activation: activated lymphocyte PKC beta may interact with insoluble cytoskeletal elements like ankyrin and spectrin. Further evidence for a link between the subcellular organization of these proteins and PKC activity is provided by the observation that inhibitors of PKC activity cause their concomitant redistribution to the cell periphery. The dynamic nature of lymphocyte ankyrin and its ability to accumulate at sites distant from the plasma membrane are properties which may be unique to the lymphocyte form of the molecule. Its colocalization with PKC beta in the lymphocyte cytoplasm, together with its redistribution in response to physiological signals, suggests that structural protein(s) may play a role in signal transduction pathways in this cell type. Our data support the conclusion that ankyrin is not solely involved in anchorage of proteins at the plasma membrane in lymphoid cells.

Protein kinase C signaling mediates a program of cell cycle withdrawal in the intestinal epithelium.

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G(0). PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21(waf1/cip1) and p27(kip1), thus targeting all of the major G(1)/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G(0) as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCalpha alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt-villus axis revealed that PKCalpha activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit-specific events in situ. Together, these data point to PKCalpha as a key regulator of cell cycle withdrawal in the intestinal epithelium.

Epithelial-mesenchymal transition (EMT) and activated extracellular signal-regulated kinase (p-Erk) in surgically resected pancreatic cancer.

BACKGROUND: EMT or transformation to the mesenchymal phenotype plays an important role in tumor invasion and metastasis. In vitro data suggest that mesenchymal transformation may correlate with the activation of PI3 kinase and Ras/Erk pathways. We investigated the expression of EMT markers (low E-cadherin, high fibronectin, and vimentin) and their association with p-Erk in resected pancreatic cancer. METHODS: Clinical data/surgical specimens from 34 consecutive pancreatic cancer patients (pts) who underwent pancreatectomy were included. Immunohistochemical staining was performed on formalin-fixed paraffin-embedded tissues using monoclonal antibodies against vimentin, fibronectin, E-cadherin, and p-Erk. The results were correlated with clinicopathological parameters and survival. Survival analysis (log-rank test, Cox proportional hazard model), categorical data analysis (Pearson's chi-square, Fisher's exact test) and Kendall's tau were performed at a significance level of 0.05. RESULTS: The patient population was formed from 13 males and 21 females, with a median age of 66 years (range 38-84 years); American Joint Committee on Cancer (AJCC) stage 1 (n = 2), 2 (n = 27), 3 (n = 5); histological grade 1 (n = 4), 2 (n = 13), 3 (n = 16), 4 (n = 1). Median survival was 15 months (95% CI: 11-24 months). Fibronectin overexpression correlated with the presence of vimentin (p = 0.0048) and activated Erk (p = 0.0264). There was a borderline association of fibronectin with worsening grade (p = 0.06). A negative association between vimentin and E-cadherin was noted (p = 0.0024). Increased fibronectin or vimentin and decreased E-cadherin correlated with poor survival. CONCLUSION: EMT is associated with poor survival in surgically resected pancreatic adenocarcinoma. A correlation between activated Erk and fibronectin was identified that may open avenues for targeted therapy for this subgroup.

Epidermal growth factor receptor-directed therapy in esophageal cancer.

Esophageal adenocarcinoma (EAC) is one of the fastest growing malignancies in the US. The long-term survival of patients with this cancer remains poor; only 25% of patients undergoing surgical excision are alive after 5 years. Multimodal programs that incorporate radiotherapy, chemotherapy and surgery for localized tumors may result in a modest survival advantage. However, significant strides in this disease can result from the inclusion of targeted therapies. The epidermal growth factor receptor (EGFR) family represents one such target and is receiving increasing attention due to the advent of specific inhibitors. Studies conducted by us and others have shown that the overexpression of EGFR family signaling intermediates is common in Barrett's esophagus and EAC. In the latter case, EGFR expression may have prognostic significance. EGFR inhibitors, including oral tyrosine kinase inhibitors and monoclonal antibodies, result in a synergistic antitumor effect with chemotherapeutic agents or with radiotherapy. Therefore, several ongoing studies include EGFR-directed therapy either alone or in combination with chemoradiotherapy for this disease. Our study of gefitinib, oxaliplatin and radiotherapy suggested that gefitinib can be safely incorporated into an oxaliplatin-based chemoradiation program for esophageal cancer, although the clinical activity of this combination is modest. Herein, we review the current literature on this subject.

Identification and characterization of human orthologues to Saccharomyces cerevisiae Upf2 protein and Upf3 protein (Caenorhabditis elegans SMG-4).

Nonsense-mediated mRNA decay (NMD), also called mRNA surveillance, is an important pathway used by all organisms that have been tested to degrade mRNAs that prematurely terminate translation and, as a consequence, eliminate the production of aberrant proteins that could be potentially harmful. In mammalian cells, NMD appears to involve splicing-dependent alterations to mRNA as well as ribosome-associated components of the translational apparatus. To date, human (h) Upf1 protein (p) (hUpf1p), a group 1 RNA helicase named after its Saccharomyces cerevisiae orthologue that functions in both translation termination and NMD, has been the only factor shown to be required for NMD in mammalian cells. Here, we describe human orthologues to S. cerevisiae Upf2p and S. cerevisiae Upf3p (Caenorhabditis elegans SMG-4) based on limited amino acid similarities. The existence of these orthologues provides evidence for a higher degree of evolutionary conservation of NMD than previously appreciated. Interestingly, human orthologues to S. cerevisiae Upf3p (C. elegans SMG-4) derive from two genes, one of which is X-linked and both of which generate multiple isoforms due to alternative pre-mRNA splicing. We demonstrate using immunoprecipitations of epitope-tagged proteins transiently produced in HeLa cells that hUpf2p interacts with hUpf1p, hUpf3p-X, and hUpf3p, and we define the domains required for the interactions. Furthermore, we find by using indirect immunofluorescence that hUpf1p is detected only in the cytoplasm, hUpf2p is detected primarily in the cytoplasm, and hUpf3p-X localizes primarily to nuclei. The finding that hUpf3p-X is a shuttling protein provides additional indication that NMD has both nuclear and cytoplasmic components.

Role of Akt2 in regulation of metastasis suppressor 1 expression and colorectal cancer metastasis.

Survival signaling is critical for the metastatic program of cancer cells. The current study investigated the role of Akt survival proteins in colorectal cancer (CRC) metastasis and explored potential mechanisms of Akt-mediated metastasis regulation. Using an orthotopic implantation model in mice, which uniquely recapitulates the entire multistep process of CRC metastasis, combined with an inducible system of short hairpin RNA-mediated Akt isoform knockdown in human CRC cells, our studies confirm a role of Akt2 in CRC cell dissemination to distant organs in vivo. Akt2 deficiency profoundly inhibited the development of liver lesions in mice, whereas Akt1 had no effect under the experimental conditions used in the study. Array analysis of human metastatic genes identified the scaffolding protein metastasis suppressor 1 (MTSS1) as a novel Akt2-regulated gene. Inducible loss of Akt2 in CRC cells robustly upregulated MTSS1 at the messenger RNA and protein level, and the accumulated protein was functionally active as shown by its ability to engage an MTSS1-Src-cortactin inhibitory axis. MTSS1 expression led to a marked reduction in levels of functional cortacin (pcortactin Y421), an actin nucleation-promoting factor that has a crucial role in cancer cell invasion and metastasis. MTSS1 was also shown to mediate suppressive effects of Akt2 deficiency on CRC cell viability, survival, migration and actin polymerization in vitro. The relevance of these findings to human CRC is supported by analysis of The Cancer Genome Atlas (TCGA) and NCBI GEO data sets, which demonstrated inverse changes in expression of Akt2 and MTSS1 during CRC progression. Taken together, the data identify MTSS1 as a new Akt2-regulated gene, and point to suppression of MTSS1 as a key step in the metastasis-promoting effects of Akt2 in CRC cells.

Interleukin 15 offers selective protection from irinotecan-induced intestinal toxicity in a preclinical animal model.

Irinotecan (CPT-11) is a chemotherapeutic agent that is active in the treatment of a variety of solid tumor malignancies. Diarrhea represents the most common dose-limiting toxicity that is independent of the schedule of administration. A rat model with dose-limiting toxicity profiles that are similar to those observed in patients treated with CPT-11 was developed and used to evaluate the role of interleukin 15 (IL-15) in the modulation of the therapeutic selectivity of CPT-11 in normal rats and rats bearing advanced colorectal cancer. The maximum tolerated dose and lethal dose (LD) of CPT-11 by i.v. push daily x 3 were 150 and 200 mg/kg/day, respectively. CPT-11 at the LD induced a 93-100% incidence of severe diarrhea and an 86-100% incidence of lethality in treated animals. IL-15, a cytokine with multiple mechanisms of action, was used at a 100 or 400 microg/kg/dose with different schedules of administration (3, 8, and 11 doses, i.p.) to protect against CPT-11-induced toxicity. IL-15 offered complete and sustained selective protection against CPT-11-induced delayed diarrhea and lethality. IL-15 also moderately potentiated the antitumor activity of CPT-11 in rats bearing advanced colorectal cancer. Morphological examination of rat intestinal tissues after treatment with LD of CPT-11 revealed dramatic protection of duodenal and colonic tissue architecture by IL-15. CPT-11 alone produced serious damage to duodenal villi and colonic crypts. The results clearly demonstrated the ability of IL-15 to provide significant protection from CPT-11-induced intestinal toxicity with maintenance of antitumor activity, resulting in an increase in the therapeutic index of CPT-11. The clinical relevance of the results obtained in this model system needs to be confirmed.

Inhibition of messenger RNA transcriptional activity in ML-1 human myeloblastic leukemia cell nuclei by antiserum to a c-myb-specific peptide.

Antiserum to a synthetic peptide that defines a hydrophilic region within the putative c-myb translation product was prepared in the rabbit. In lysates from exponentially growing ML-1, human myeloblastic leukemia cells, the antiserum ("anti-myb") reacted with five proteins of Mr 58,000, 75,000, 85,000, 90,000 and 105,000. Of these, only p75 and a trace of p85 were detected, by immunoblotting, in extracts derived from ML-1 cell nuclei. The proteins p58, p75 and p90 were present in readily detectable amounts only in the relatively immature myeloid cell lines ML-1 and HL-60, whereas in the more mature myeloid cell line THP-1 and in the lymphoid line BALL-1 only traces of these proteins were found. p85 and p105 were detected in lysates from all cell lines tested, including myeloid and lymphoid leukemia cells and mouse 3T3 cells. In lysates from ML-1 cells induced to differentiate to monocyte/macrophages or to granulocytes, the concentrations of p58 and p75 decreased in parallel with the cell population moving to maturity; in completely mature populations these two proteins were no longer detectable. In ML-1 cells arrested in G1 by serum depletion, the amount of p58 and p75 and to a smaller extent that of p90 was decreased, whereas the concentration of p85 and p105 remained unchanged. In nuclei from exponentially growing ML-1 cells, the antiserum or its derived immunoglobulin fraction ("anti-myb IgG") inhibited mRNA transcriptional activity by 30%. DNA synthesis was not affected. In contrast, in nuclei from differentiated ML-1 cells, the mRNA transcriptional activity was not significantly inhibited by anti-myb IgG. Similarly, in nuclei from ML-1 cells arrested largely in G1 by serum depletion for 2 days, mRNA transcriptional activity was inhibited by only 11%. Upon supplementation with serum, the mRNA transcriptional activity inhibitable by anti-myb IgG increased in parallel with the increasing rate of cell growth. The difference in total mRNA transcriptional activity observed in nuclei from cells of different growth rate was accounted for by the difference in transcriptional activity inhibitable by anti-myb IgG.(ABSTRACT TRUNCATED AT 400 WORDS)

Lysosomal sequestration of polyamine analogues in Chinese hamster ovary cells resistant to the S-adenosylmethionine decarboxylase inhibitor, CGP-48664.

CGP-48664, an inhibitor of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC), is presently undergoing Phase 1 clinical trials as an experimental anticancer agent. We have shown previously (D. L. Kramer et al., J. Biol. Chem., 270: 2124-2132, 1995) that Chinese hamster ovary (CHO) cells that are made resistant to the growth inhibitory effects of the drug overexpress AdoMetDC because of a stable gene amplification. Unexpectedly, these same cells (CHO/644) were found to be insensitive to the growth inhibitory effects of N1,N11-diethylnorspermine (DENSPM)-a polyamine analogue also undergoing Phase 1 clinical trials-despite accumulating approximately 5 times more analogue than parental cells. We now report that treatment of CHO/664 cells with DENSPM results in the formation of numerous large cytoplasmic vacuoles, which on the basis of electron microscopy and cytochemical staining seem to be lysosomal in origin. A series of newly established CHO cell lines made differentially resistant to 1, 3, 10, 30, and 100 microM CGP-48664 by chronic exposure were used to demonstrate that vacuole formation correlated with the accumulation of extremely high levels of DENSPM without increasing growth inhibition. These same cells were used to show that AdoMetDC gene overexpression as indicated by mRNA levels was unrelated to vacuole formation; cells resistant to 100 microM CGP-48664 displayed a 170-fold increase in AdoMetDC mRNA levels and formed vacuoles in response to DENSPM, whereas those resistant to 10 microM CGP-48664 displayed a 120-fold increase in AdoMetDC mRNA levels and failed to form vacuoles. Despite accumulating to high intracellular levels, DENSPM was much less effective than spermine at down-regulating ornithine decarboxylase and polyamine transport activities in highly resistant cells. Similarly, DENSPM was less able to induce spermidine/spermine N1-acetyltransferase activity in cells that formed vacuoles than in those that did not. Overall, natural polyamines failed to induce vacuoles and various analogues of DENSPM were used to probe the structural specificity of the effect. The data are consistent with the probability that DENSPM is sequestered to high concentrations in lysosomal vacuoles of CGP-48664-resistant cells and is, therefore, not available to interact with polyamine regulatory sites or to cytotoxically affect cell growth. In addition to implicating the lysosome as a potential new site of CGP-48664 drug action that could be involved in antitumor activity and/or host toxicities, the findings also suggest a potential mechanism of cell resistance to analogues such as DENSPM.

Polyamine analogue induction of the p53-p21WAF1/CIP1-Rb pathway and G1 arrest in human melanoma cells.

Although polyamines are well recognized for their critical involvement in cell growth, the cell cycle specificity of this requirement has not yet been characterized with respect to the newly delineated regulatory pathways. We recently reported that polyamine analogues having close structural and functional similarities to the natural polyamines produce a distinct G1 and G2-M cell cycle arrest in MALME-3M human melanoma cells. To determine a molecular basis for this observation, we examined the effects of N1,N11-diethylnorspermine on cell cycle regulatory proteins associated with G1 arrest. The analogue is known to deplete polyamine pools by suppressing biosynthetic enzymes and potently inducing the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase. Treatment of MALME-3M cells with 10 microM N1,N11-diethylnorspermine caused an increase in hypophosphorylated Rb, which correlated temporally with the onset of G1 arrest at 16-24 h. Rb hypophosphorylation was preceded by an increase in wild-type p53 (approximately 100-fold at maximum) and a concomitant increase in the cyclin-dependent kinase inhibitor, p21WAF1/CIP1 (p21; approximately 5-fold at maximum). Another cyclin-dependent kinase inhibitor, p27KIP1, and cyclin D increased slightly, whereas proliferating cell nuclear antigen and p130 remained unchanged. Induction of p21 protein was accompanied by an increase in p21 mRNA, whereas induction of p53 protein was not, suggesting transcriptional activation of the former and posttranscriptional regulation of the latter. SK-MEL-28 human melanoma cells, which contain a mutated p53, failed to induce p53 or p21 and did not arrest in G1. Rather, these cells rapidly underwent programmed cell death within 48 h. Overall, these findings provide the first indication of the cell cycle regulatory pathways by which polyamine antagonists such as analogues might inhibit growth in cells containing wild-type p53 and further suggest a mechanistic basis for differential cellular responses to these agents.

Polarized expression of immunoglobulin, spectrin, and protein kinase C beta II occurs in B cells from normal BALB/c, autoimmune lpr, and anti-ssDNA transgenic, tolerant mice.

The rapid redistribution of B cell surface immunoglobulin to a cap upon cross-linking treatment is a well-described phenomenon, the physiological significance of which is unknown. We describe the observation that splenic B cells from unimmunized normal, autoimmune, and tolerant mice express naturally occurring capped immunoglobulin in the absence of exogenous stimulation. The percentage of capped B cells increases to 20% of B cells by age 16 weeks in the progressive autoimmune lpr mouse. Transgenic, tolerant mice expressing lpr-derived genes for ssDNA-binding antibody also demonstrate a large percentage (35-75%) of immunoglobulin-capped splenic B cells. In these capped B cells, protein kinase C beta II, the cytoskeletal proteins spectrin and ankyrin, and the lipophilic probe diI are enriched beneath the site of the immunoglobulin cap. These data suggest that polarization of surface receptors, signaling molecules, anionic phospholipid domains, and cytoskeletal proteins may be an important part of the B cell immune response in vivo.

Familial and nutritional risk factors for p53 overexpression in colorectal cancer.

Although familial and dietary factors are recognized as important risk determinants for colorectal tumorigenesis, the specific causes of colorectal cancer remain unclear. Studies of p53 genetic alterations have provided clues concerning the etiology of many cancers. This study was designed to determine whether overexpression of the p53 protein is associated with familial and dietary risk factors. Epidemiological data were obtained from 163 colorectal cancer cases and 326 healthy controls. Tumors of all patients were analyzed immunohistochemically for p53 overexpression using an avidin-biotin immunoperoxidase procedure and polyclonal anti-p53 antibody CM1. Of patient tumors, 44.8% showed p53 nuclear reactivity. Colorectal cases versus controls were three times more likely to report a family history of colorectal cancer [odds ratio (OR), 3.12; 95% confidence interval (CI), 1.77-5.52]. Only cruciferous vegetables exhibited a significant inverse association (OR, 0.59; 95% CI, 0.34-1.02; trend test, P = 0.03) for the highest versus lowest quartiles. Both meat and beef displayed an elevated increase in risk. When cases with p53 overexpression (p53 positive) were compared with cases without p53 overexpression (p53 negative), etiological heterogeneity was suggested for family history of colorectal cancer (OR, 0.39; 95% CI, 0.16-0.93), cruciferous vegetables (trend test, P = 0.12), and beef consumption (trend test, P = 0.08). To estimate the individual relative risks for p53-dependent and p53-independent pathways, each p53 subtype was compared with controls. Cruciferous vegetables exhibited a significant association (OR, 0.37; 95% CI, 0.17-0.82; trend test, P = 0.03) when p53 positive cases were compared with controls. When p53 negative cases were compared with controls, a significant increase in risk was observed for family history of cancer (OR, 4.46; 95% CI, 2.36-8.43) and beef (OR, 3.17; 95% CI, 1.83-11.28; trend test, P = 0.006). The p53 (positive) dependent pathway was characterized by an inverse association with cruciferous vegetables, and p53-independent tumors were characterized by family history and beef consumption. These data may indicate the significance of linking epidemiology and molecular biology in assessing specific etiological pathways.

Protein kinase C-mediated regulation of the cell cycle.

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth/cell cycle progression and differentiation. Increasing evidence from studies using in vitro and in vivo systems points to PKC as a key regulator of critical cell cycle transitions, including cell cycle entry and exit and the G1 and G2 checkpoints. PKC-mediated control of these transitions can be negative or positive, depending on the timing of PKC activation during the cell cycle and on the specific PKC isozymes involved. Most of the mechanistic information available relates to the involvement of this enzyme family in negative regulation of these transitions. Accumulating data indicate that a major target for PKC-mediated inhibition of cell cycle progression is the Cip/Kip cyclin-dependent kinase (cdk) inhibitor p21waf1/cip1. Increased expression of p21waf1/cip1 blocks cdk2 activity in G1 phase, leading to hypophosphorylation of the retinoblastoma protein and inhibition of cell cycle progression into S phase. In G2, p21waf1/cip1 expression blocks cdc2/cyclin B activity, likely through an indirect mechanism involving inhibition of the cdk2/cyclin A complex, and prevents progression into M phase. PKC signaling can also activate a coordinated program of pocket protein regulation leading to cell cycle withdrawal into G0. The molecular events underlying positive regulation of cell cycle progression by PKC signaling remain poorly understood, although there is evidence for a role of the enzyme in promoting G2(r)M progression by phosphorylating lamin B at sites involved in nuclear lamina disassembly. Understanding of the mechanisms underlying PKC-mediated control of the cell cycle is beginning to provide important insight into its role in uncontrolled cell growth and transformation.

Selective location of acceptors for botulinum neurotoxin A in the central and peripheral nervous systems.

The main site of action for botulinum neurotoxin is cholinergic motor nerve terminals where specific acceptors concentrate the toxin on the cell surface, thereby facilitating its internalization and inactivation of a component essential for transmitter release. In this study, the interaction in vitro of [125I]botulinum neurotoxin type A with central and peripheral nerve terminals of different types was investigated using Ultrofilm and electron-microscope autoradiography. It was found that: (i) The neurotoxin binds to synapse-rich areas of rat brain, particularly in the hippocampus and cerebellum; identity of the neuron types labelled is unclear although cholinergic nerves seem to be labelled, perhaps not exclusively, in many areas. (ii) Toxin uptake at central nerve terminals appears to be minimal and its penetration into intact brain slices is restricted; this may account for the toxin's lower central toxicity. (iii) Selective labelling of cholinergic nerves but not purinergic, peptidergic or adrenergic nerve terminals in mouse ileum suggests that the toxin may be a specific marker for cholinergic nerves in the periphery. Based on these localization studies and published pharmacological observations, it is concluded that efficient toxin-induced blockade of neurotransmission depends on the presence of specific acceptors of high affinity for the toxin and of an effective neuronal uptake mechanism. Inhibition of the release of numerous transmitters from different kinds of nerve terminals lacking one of these features can be produced by high toxin concentrations when uptake occurs via low affinity acceptors or by non-specific means. Notably, this widespread action of the toxin indicates the occurrence of a common intracellular target in several, possibly all, nerve types.

Stimulation of protein kinase C-dependent and -independent signaling pathways by bistratene A in intestinal epithelial cells.

The marine toxin bistratene A (BisA) potently induces cytostasis and differentiation in a variety of systems. Evidence that BisA is a selective activator of protein kinase C (PKC) delta implicates PKC delta signaling in the negative growth-regulatory effects of this agent. The current study further investigates the signaling pathways activated by BisA by comparing its effects with those of the PKC agonist phorbol 12-myristate 13-acetate (PMA) in the IEC-18 intestinal crypt cell line. Both BisA and PMA induced cell cycle arrest in these cells, albeit with different kinetics. While BisA produced sustained cell cycle arrest in G(0)/G(1) and G(2)/M, the effects of PMA were transient and involved mainly a G(0)/G(1) blockade. BisA also produced apoptosis in a proportion of the population, an effect not seen with PMA. Both agents induced membrane translocation/activation of PKC, with BisA translocating only PKC delta and PMA translocating PKC alpha, delta, and epsilon in these cells. Notably, while depletion of PKC alpha, delta, and epsilon abrogated the cell cycle-specific effects of PMA in IEC-18 cells, the absence of these PKC isozymes failed to inhibit BisA-induced G(0)/G(1) and G(2)/M arrest or apoptosis. The cell cycle inhibitory and apoptotic effects of BisA, therefore, appear to be PKC-independent in IEC-18 cells. On the other hand, BisA and PMA both promoted PKC-dependent activation of Erk 1 and 2 in this system. Thus, intestinal epithelial cells respond to BisA through activation of at least two signaling pathways: a PKC delta-dependent pathway, which leads to activation of mitogen-activated protein kinase and possibly cytostasis in the appropriate context, and a PKC-independent pathway, which induces both cell cycle arrest in G(0)/G(1) and G(2)/M and apoptosis through as yet unknown mechanisms.