The onset and extent of genomic instability in sporadic colorectal tumor progression

Cancer cell genomes contain alterations beyond known etiologic events, but their total number has been unknown at even the order of magnitude level. By sampling colorectal premalignant polyp and carcinoma cell genomes through use of the technique inter-(simple sequence repeat) PCR, we have found genomic alterations to be considerably more abundant than expected, with the mean number of genomic events per carcinoma cell totaling approximately 11,000. Colonic polyps early in the tumor progression pathway showed similar numbers of events. These results indicate that, as with certain hereditary cancer syndromes, genomic destabilization is an early step in sporadic tumor development. Together these results support the model of genomic instability being a cause rather than an effect of malignancy, facilitating vastly accelerated somatic cell evolution, with the observed orderly steps of the colon cancer progression pathway reflecting the consequences of natural selection.

Lipotoxic heart disease in obese rats: Implications for human obesity

To determine the mechanism of the cardiac dilatation and reduced contractility of obese Zucker Diabetic Fatty rats, myocardial triacylglycerol (TG) was assayed chemically and morphologically. TG was high because of underexpression of fatty acid oxidative enzymes and their transcription factor, peroxisome proliferator-activated receptor-α. Levels of ceramide, a mediator of apoptosis, were 2–3 times those of controls and inducible nitric oxide synthase levels were 4 times greater than normal. Myocardial DNA laddering, an index of apoptosis, reached 20 times the normal level. Troglitazone therapy lowered myocardial TG and ceramide and completely prevented DNA laddering and loss of cardiac function. In this paper, we conclude that cardiac dysfunction in obesity is caused by lipoapoptosis and is prevented by reducing cardiac lipids.

Mutated epithelial cadherin is associated with increased tumorigenicity and loss of adhesion and of responsiveness to the motogenic trefoil factor 2 in colon carcinoma cells

Epithelial (E)-cadherin and its associated cytoplasmic proteins (α-, β-, and γ-catenins) are important mediators of epithelial cell–cell adhesion and intracellular signaling. Much evidence exists suggesting a tumor/invasion suppressor role for E-cadherin, and loss of expression, as well as mutations, has been described in a number of epithelial cancers. To investigate whether E-cadherin gene (CDH1) mutations occur in colorectal cancer, we screened 49 human colon carcinoma cell lines from 43 patients by single-strand conformation polymorphism (SSCP) analysis and direct sequencing. In addition to silent changes, polymorphisms, and intronic variants in a number of the cell lines, we detected frameshift single-base deletions in repeat regions of exon 3 (codons 120 and 126) causing premature truncations at codon 216 in four replication-error-positive (RER+) cell lines (LS174T, HCT116, GP2d, and GP5d) derived from 3 patients. In LS174T such a mutation inevitably contributes to its lack of E-cadherin protein expression and function. Transfection of full-length E-cadherin cDNA into LS174T cells enhanced intercellular adhesion, induced differentiation, retarded proliferation, inhibited tumorigenicity, and restored responsiveness to the migratory effects induced by the motogenic trefoil factor 2 (human spasmolytic polypeptide). These results indicate that, although inactivating E-cadherin mutations occur relatively infrequently in colorectal cancer cell lines overall (3/43 = 7%), they are more common in cells with an RER+ phenotype (3/10 = 30%) and may contribute to the dysfunction of the E-cadherin–catenin-mediated adhesion/signaling system commonly seen in these tumors. These results also indicate that normal E-cadherin-mediated cell adhesion can restore the ability of colonic tumor cells to respond to trefoil factor 2.

The adenomatous polyposis coli-binding protein EB1 is associated with cytoplasmic and spindle microtubules

The evolutionarily conserved protein EB1 originally was identified by its physical association with the carboxyl-terminal portion of the adenomatous polyposis coli (APC) tumor suppressor protein, an APC domain commonly mutated in familial and sporadic forms of colorectal neoplasia. The subcellular localization of EB1 in epithelial cells was studied by using immunofluorescence and biochemical techniques. EB1 colocalized both to cytoplasmic microtubules in interphase cells and to spindle microtubules during mitosis, with pronounced centrosome staining. The cytoskeletal array detected by anti-EB1 antibody was abolished by incubation of the cells with nocodazole, an agent that disrupts microtubules; upon drug removal, EB1 localized to the microtubule-organizing center. Immunofluorescence analysis of SW480, a colon cancer cell line that expresses only carboxyl-terminal-deleted APC unable to interact with EB1, demonstrated that EB1 remained localized to the microtubule cytoskeleton, suggesting that this pattern of subcellular distribution is not mediated by its interaction with APC. In vitro cosedimentation with taxol-stabilized microtubules demonstrated that a significant fraction of EB1 associated with microtubules. Recent studies of the yeast EB1 homologues Mal3 and Bim1p have demonstrated that both proteins localize to microtubules and are important in vivo for microtubule function. Our results demonstrate that EB1 is a novel component of the microtubule cytoskeleton in mammalian cells. Associating with the mitotic apparatus, EB1 may play a physiologic role connecting APC to cellular division, coordinating the control of normal growth and differentiation processes in the colonic epithelium.

Supraspinal contributions to hyperalgesia

Tissue injury is associated with sensitization of nociceptors and subsequent changes in the excitability of central (spinal) neurons, termed central sensitization. Nociceptor sensitization and central sensitization are considered to underlie, respectively, development of primary hyperalgesia and secondary hyperalgesia. Because central sensitization is considered to reflect plasticity at spinal synapses, the spinal cord has been the principal focus of studies of mechanisms of hyperalgesia. Not surprisingly, glutamate, acting at a spinal N-methyl-d-aspartate (NMDA) receptor, has been implicated in development of secondary hyperalgesia associated with somatic, neural, and visceral structures. Downstream of NMDA receptor activation, spinal nitric oxide (NO⋅), protein kinase C, and other mediators have been implicated in maintaining such hyperalgesia. Accumulating evidence, however, reveals a significant contribution of supraspinal influences to development and maintenance of hyperalgesia. Spinal cord transection prevents development of secondary, but not primary, mechanical and/or thermal hyperalgesia after topical mustard oil application, carrageenan inflammation, or nerve-root ligation. Similarly, inactivation of the rostral ventromedial medulla (RVM) attenuates hyperalgesia and central sensitization in several models of persistent pain. Inhibition of medullary NMDA receptors or NO⋅ generation attenuates somatic and visceral hyperalgesia. In support, topical mustard oil application or colonic inflammation increases expression of NO⋅ synthase in the RVM. These data suggest a prominent role for the RVM in mediating the sensitization of spinal neurons and development of secondary hyperalgesia. Results to date suggest that peripheral injury and persistent input engage spinobulbospinal mechanisms that may be the prepotent contributors to central sensitization and development of secondary hyperalgesia.

On the etiology of Crohn disease.

Crohn disease (CD) is a chronic, panenteric intestinal inflammatory disease. Its etiology is unknown. Analogous to the tuberculoid and lepromatous forms of leprosy, CD may have two clinical manifestations. One is aggressive and fistulizing (perforating), and the other is contained, indolent, and obstructive (nonperforating) [Gi]-berts, E. C. A. M., Greenstein, A. J., Katsel, P., Harpaz, N. & Greenstein, R. J. (1994) Proc. Natl. Acad. Sci. USA 91, 12721-127241. The etiology, if infections, may be due to Mycobacterium paratuberculosis. We employed reverse transcription PCR using M. paratuberculosis subspecies-specific primers (IS 900) on total RNA from 12 ileal mucosal specimens (CD, n = 8; controls, n = 4, 2 with ulcerative colitis and 2 with colonic cancer). As a negative control, we used Myobacterium avium DNA, originally cultured from the drinking water of a major city in the United States. cDNA sequence analysis shows that all eight cases of Crohn's disease and both samples from the patients with ulcerative colitis contained M. paratuberculosis RNA. Additionally, the M. avium control has the DNA sequence of M. paratuberculosis. We demonstrate the DNA sequence of M. paratuberculosis from mucosal specimens from humans with CD. The potable water supply may be a reservoir of infection. Although M. paratuberculosis signal in CD has been previously reported, a cause and effect relationship has not been established. In part, this is due to conflicting data from studies with empirical antimycobacterial therapy. We conclude that clinical trials with anti-M. paratuberculosis therapy are indicated in patients with CD who have been stratified into the aggressive (perforating) and contained (nonperforating) forms.

Receptor-specific in vivo desensitization by the G protein-coupled receptor kinase-5 in transgenic mice.

Transgenic mice were generated with cardiac-specific overexpression of the G protein-coupled receptor kinase-5 (GRK5), a serine/threonine kinase most abundantly expressed in the heart compared with other tissues. Animals overexpressing GRK5 showed marked beta-adrenergic receptor desensitization in both the anesthetized and conscious state compared with nontransgenic control mice, while the contractile response to angiotensin II receptor stimulation was unchanged. In contrast, the angiotensin II-induced rise in contractility was significantly attenuated in transgenic mice overexpressing the beta-adrenergic receptor kinase-1, another member of the GRK family. These data suggest that myocardial overexpression of GRK5 results in selective uncoupling of G protein-coupled receptors and demonstrate that receptor specificity of the GRKs may be important in determining the physiological phenotype.

Toward antibody-directed "abzyme" prodrug therapy, ADAPT: carbamate prodrug activation by a catalytic antibody and its in vitro application to human tumor cell killing.

Antibody-directed enzyme prodrug therapy, ADEPT, is a recent approach to targeted cancer chemotherapy intended to diminish the nonspecific toxicity associated with many commonly used chemotherapeutic agents. Most ADEPT systems incorporate a bacterial enzyme, and thus their potential is reduced because of the immunogenicity of that component of the conjugate. This limitation can be circumvented by the use of a catalytic antibody, which can be "humanized," in place of the bacterial enzyme catalyst. We have explored the scope of such antibody-directed "abzyme" prodrug therapy, ADAPT, to evaluate the potential for a repeatable targeted cancer chemotherapy. We report the production of a catalytic antibody that can hydrolyze the carbamate prodrug 4-[N,N-bis(2-chloroethyl)]aminophenyl-N-[(1S)-(1,3- dicarboxy)propyl]carbamate (1) to generate the corresponding cytotoxic nitrogen mustard (Km = 201 microM, kcat = 1.88 min-1). In vitro studies with this abzyme, EA11-D7, and prodrug 1 lead to a marked reduction in viability of cultured human colonic carcinoma (LoVo) cells relative to appropriate controls. In addition, we have found a good correlation between antibody catalysis as determined by this cytotoxicity assay in vitro and competitive binding studies of candidate abzymes to the truncated transition-state analogue ethyl 4-nitrophenylmethylphosphonate. This cell-kill assay heralds a general approach to direct and rapid screening of antibody libraries for catalysts.

Identification of the galactose-adherence lectin epitopes of Entamoeba histolytica that stimulate tumor necrosis factor-alpha production by macrophages.

The 170-kDa subunit of the galactose-adherence lectin (Gal-lectin) of Entamoeba histolytica mediates adherence to human colonic mucins and intestinal epithelium as a prerequisite to amebic invasion. The Gal-lectin is an immunodominant molecule and a protective antigen in the gerbil model of amebiasis. Tumor necrosis factor alpha (TNF-alpha) produced by activated macrophages enhances nitric oxide-dependent cytotoxicity in host defense against E. histolytica. The purpose of this study was to identify the Gal-lectin epitopes which stimulate TNF-alpha production by macrophages. Murine bone marrow-derived macrophages (BMMs) exposed to Gal-lectin (100-500 ng/ml) stimulated stable expression of TNF-alpha mRNA (8-fold increase) and TNF-alpha production similar to that of lipopolysaccharide-stimulated cells (100 ng/ml). Polyclonal anti-lectin serum specifically inhibited TNF-alpha mRNA induction in response to the Gal-lectin but not to lipopolysaccharide. Anti-lectin monoclonal antibodies 8C12, H85 and 1G7, which recognize nonoverlapping epitopes of the cysteine-rich region of the 170-kDa heavy subunit, inhibited both amebic adherence to mammalian cells and Gal-lectin-stimulated TNF-alpha mRNA expression by BMMs,but monoclonal antibody 7F4 did neither. As these inhibitory antibodies map to amino acids 596-1082 of the 170-kDa Gal-lectin, our results have identified the functional region that mediates amebic adherence and TNF-alpha mRNA induction in BMMMs; thus, this region of the Gal-lectin is a subunit vaccine candidate.

Increased expression and activity of sodium channels in alveolar type II cells of hyperoxic rats.

We investigated the cellular and molecular events associated with the increase in sodium transport across the alveolar epithelium of rats exposed to hyperoxia (85% O2 for 7 days followed by 100% O2 for 4 days). Alveolar type II (ATII) cell RNA was isolated and probed with a cDNA for one of the rat colonic epithelial sodium channel subunits (alpha rENaC). The alpha rENaC mRNA (3.7-kb transcript) increased 3-fold in ATII cell RNA isolated from rats exposed to 85% O2 for 7 days and 6-fold after 4 days of subsequent exposure to 100% O2. In situ hybridization revealed increased expression of alpha rENaC mRNA transcripts in both airway and alveolar epithelial cells of hyperoxic rats. When immunostained with a polyclonal antibody to kidney sodium channel protein, ATII cells from hyperoxic rats exhibited a significant increase in the amount of immunogenic protein present in both the plasma membrane and the cytoplasm. When patched in the whole-cell mode, ATII cells from hyperoxic rats exhibited amiloride and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride (EIPA)-sensitive currents that were 100% higher compared with those obtained from air-breathing rats. Single-channel sodium currents (mean conductance of 25 pS) were seen in ATII cells patched in both the inside-out and cell-attached modes. The number and open probability of these channels increased significantly during exposure to hyperoxia. Exposure to sublethal hyperoxia up-regulated both alpha rENaC mRNA and the functional expression of sodium channels in ATII cells.

Contrasting roles for Myc and Mad proteins in cellular growth and differentiation.

The positive effects of Myc on cellular growth and gene expression are antagonized by activities of another member of the Myc superfamily, Mad. Characterization of the mouse homolog of human mad on the structural level revealed that domains shown previously to be required in the human protein for anti-Myc repression, sequence-specific DNA-binding activity, and dimerization with its partner Max are highly conserved. Conservation is also evident on the biological level in that both human and mouse mad can antagonize the ability of c-myc to cooperate with ras in the malignant transformation of cultured cells. An analysis of c-myc and mad gene expression in the developing mouse showed contrasting patterns with respect to tissue distribution and developmental stage. Regional differences in expression were more striking on the cellular level, particularly in the mouse and human gastrointestinal system, wherein c-Myc protein was readily detected in immature proliferating cells at the base of the colonic crypts, while Mad protein distribution was restricted to the postmitotic differentiated cells in the apex of the crypts. An increasing gradient of Mad was also evident in the more differentiated subcorneal layers of the stratified squamous epithelium of the skin. Together, these observations support the view that both downregulation of Myc and accumulation of Mad may be necessary for progression of precursor cells to a growth-arrested, terminally differentiated state.