Inhibition of epidermal growth factor receptor tyrosine kinase fails to suppress adenoma formation in ApcMin mice but induces duodenal injury.

A highly selective, p.o. bioavailable irreversible inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, N-[4-(3-chloro4-fluorophenylamino)-quinazolin-6-yl]-ac rylamide (CFPQA), was evaluated for its ability to prevent intestinal adenoma formation in ApcMin mice. Ten-week continuous dietary exposure to CFPQA at doses sufficient to abolish intestinal EGFR tyrosine phosphorylation failed to affect intestinal tumor multiplicity or distribution but induced flat mucosal lesions in the duodenum characteristic of chronic injury. Intestinal trefoil factor, an intestinal peptide that mediates antiapoptotic effects through an EGFR-dependent mechanism, was notably absent in adenomas but was highly expressed in flat duodenal lesions. We conclude that chronic inhibition of EGFR tyrosine kinase by CFPQA does not prevent adenomas in ApcMin mice but may induce duodenal injury.

Adenoma-specific alterations of protein kinase C isozyme expression in Apc(MIN) mice.

Members of the protein kinase C (PKC) family appear to play important roles in colorectal carcinogenesis. To investigate the potential involvement of PKC isozymes in adenomatous transformation induced by inactivation of the adenomatous polyposis coli (APC) gene product, we examined protein levels and localizations of ten PKC isozymes by immunohistochemistry in normal and adenomatous ileal epithelium of ApcMIN mice. Compared with surrounding normal epithelium, adenomas showed dramatically reduced staining for PKCs a, beta1, and zeta, as well as dysplasia-specific punctate nuclear staining of PKC mu. We conclude that reduced protein expression of PKC alpha, beta1, and zeta, and nuclear localization of PKC mu are markers of, and are perhaps involved in, adenomatous transformation induced by APC inactivation in ApcMIN mice.

Implications of low COX-2 expression in colorectal neoplasms with defective DNA mismatch repair.

Most colorectal adenomas and carcinomas arise in the setting of chromosomal instability and progressive loss of heterozygosity. Approximately 15-20% of colorectal neoplasms arise through a distinct genetic pathway characterized by microsatellite instability (MSI) and frequent loss of expression of one of the DNA mismatch repair enzymes, most often hMLH1 or hMSH2. These distinct genetic pathways are reflected by differences in tumor histopathology, distribution in the colon, prognosis, and dwell time required for progression from adenoma to carcinoma. The molecular and clinical distinctions between these tumors suggest that they are biologically distinct and may respond differently to therapeutic and chemopreventive agents. Recently, we showed that expression of a putative chemopreventive target, COX-2, is significantly reduced in colorectal cancers with defective mismatch repair as assessed by MSI and absent staining for hMLH1 and/or hMSH2. The mechanisms responsible for low COX-2 expression in tumors with MSI remain unknown, but they may be linked to molecular events giving rise to MSI tumors. Although the clinical implications of these observations are unknown, the presence of MSI should be considered an important variable when assessing the efficacy of COX-2 inhibitors in chemoprevention trials.

Distinct protein kinase C isozymes signal mitogenesis and apoptosis in human colon cancer cells.

BACKGROUND & AIMS: Protein kinase C (PKC) is a family of serine-threonine kinases that transmit signals from cell surface receptors. To determine if distinct PKC isozymes transmit proliferative and/or apoptotic signals in colon cancer cells, we examined the effects of 3 PKC agonists, phorbol 12-myristate 13 acetate (PMA), ingenol 3,20-dibenzoate (IDB), and bistratene A, and a selective PKC inhibitor, GF 109203X, on proliferation, apoptosis, and activation of individual PKC isozymes in 5 colon cancer cell lines. METHODS: Effects were assayed by a formazan-based colorimetric assay, [(3)H]thymidine incorporation, fluorescent nuclear staining, annexin V binding, DNA fragmentation assay, and immunoblotting of cytoplasmic and membrane fractions for PKC isozymes. RESULTS: Two cell lines, SNU-C1 and SNU-C4, showed proliferative responses to PMA (0.1-1 nmol/L) and IDB (10-1000 nmol/L) and marked apoptotic responses to PMA (>5 nmol/L) and bistratene A (>1 micromol/L). GF 109203X blocked proliferative and apoptotic effects of PMA with distinct IC(50)s. Proliferative concentrations of PMA and IDB caused translocation of PKCepsilon alone, whereas apoptotic concentrations of PMA and bistratene A induced translocation of PKCdelta. CONCLUSIONS: Activation of PKCepsilon and PKCdelta triggers proliferative and apoptotic signals, respectively, in SNU-C4 colon cancer cells. These 2 isozymes may play important opposing roles in normal homeostasis and neoplastic transformation of the colorectal epithelium.

Cloning and characterization of the murine PKC alpha promoter: identification of a retinoic acid response element.

Protein kinase C (PKC) is a family which consists of multiple isoforms whose distinct physiological roles within the cell are unknown. We have previously demonstrated that levels of PKC alpha mRNA, protein, and enzyme activity in B16 melanoma cells can be modulated by retinoic acid. We investigated this regulation by cloning and characterizing the promoter region of the murine PKC alpha gene. A 13 kb mouse genomic fragment containing the 5' flanking region, first exon, and first intron was isolated and sequenced. Two transcription initiation sites were identified at 919 and 925 bp upstream from the translation start site. The promoter region contained a TATA-like box at -93 bp upstream of the transcription start site, but no CAAT box. Promoter activity differed between cell lines and correlated with the levels of PKC alpha expressed in these cell lines. Reporter gene assays showed that the region between -179 and -452 bp likely contains a silencer element(s). The promoter activity of a -179 bp fragment in B16 cells was stimulated twofold by retinoic acid. Within this region (-93 to -65 bp) there is a retinoic acid response element. An oligonucleotide spanning this region specifically bound exogenous RAR-RXR heterodimers and endogenous RAR from B16 nuclear extracts. These results suggest that retinoic acid increases PKC alpha gene expression in B16 cells, at least in part, through direct transcriptional stimulation of its promoter.

Reduced COX-2 protein in colorectal cancer with defective mismatch repair.

Most colorectal adenomas and carcinomas arise in the setting of chromosomal instability characterized by progressive loss of heterozygosity. In contrast, approximately 15-20% of colorectal neoplasms arise through a distinct genetic pathway characterized by microsatellite instability (MSI) associated with frequent loss of expression of one of the DNA mismatch repair enzymes, most often hMLH1 or hMSH2. These distinct genetic pathways are reflected by differences in tumor histopathology, distribution in the colon, prognosis, and dwell time required for progression from adenoma to carcinoma. To determine whether these two groups of tumors differ in their expression of cyclooxygenase-2 (COX-2), a putative chemopreventative target, immunostaining for this protein was performed in colorectal cancers categorized by the presence (n = 41) and absence (n = 66) of defective mismatch repair. Defective mismatch repair was defined by the presence of tumor microsatellite instability (MSI-H, > or =40% of markers demonstrating instability) and by the absence of protein expression for either hMLH1 or hMSH2. Overall, our results showed that low or absent COX-2 staining was significantly more common among tumors with defective mismatch repair (P = 0.001). Other features predictive of low COX-2 staining included marked tumor infiltrating lymphocytosis, and solid/cribiform or signet ring histological patterns. These observations indicate that colorectal cancers with molecular and phenotypic characteristics of defective DNA mismatch repair express lower levels of COX-2. The clinical implications of this biological distinction remain unknown but should be considered when assessing the efficacy of COX-2 inhibitors for chemoprevention in patients whose tumors may arise in the setting of defective DNA mismatch repair.

Inhibition of epidermal growth factor receptor kinase induces protease-dependent apoptosis in human colon cancer cells.

BACKGROUND & AIMS: The epidermal growth factor receptor (EGFR) is under investigation as a therapeutic target for cancers. Colon cancer cell lines are variably dependent on autocrine stimulation of EGFR. We therefore examined the effects of a selective EGFR tyrosine kinase inhibitor, PD 153035, on proliferation and survival of five colon cancer cell lines whose autonomous proliferation is either EGFR ligand dependent or EGFR ligand independent. METHODS: Effects of inhibitors were screened by MTS growth assays, [3H]thymidine incorporation, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay, fluorescence microscopy, immunoblotting, and in vitro protease assays. RESULTS: PD 153035 caused dose-dependent cytostasis (200 nmol/L to 1 micromol/L) and apoptosis (>10 micromol/L) in ligand-dependent cell lines and caused variable apoptosis (>10 micromol/L) but no cytostasis in ligand-independent cell lines. Apoptosis induced by 10 micromol/L PD 153035 was not associated with induction of p53 protein expression but was accompanied by activation of caspases that cleave poly(ADP-ribose) polymerase, lamin B1, and Bcl-2. Inhibition of caspase 3-like protease activity by DEVD-fluoromethylketone significantly delayed the onset of PD 153035-induced apoptosis. CONCLUSIONS: The EGFR tyrosine kinase inhibitor PD 153035 induces cytostasis and caspase-dependent apoptosis in EGFR ligand-dependent colon cancer cell lines. These observations encourage further investigation of EGFR tyrosine kinase inhibitors for treatment of colorectal neoplasms.

p53 functional loss in a colon cancer cell line with two missense mutations (218leu and 248trp) on separate alleles.

We have sequenced p53 in three colon cancer cell lines capable of autonomous proliferation. SNU-C1 and SNU-C4 cells, whose autonomous growth is dependent upon autocrine stimulation of epidermal growth factor receptor (EGFR), had wildtype p53 sequence of exons 4-9. In contrast, an EGFR ligand-independent cell line, SNU-C5, had heterozygous missense mutations affecting codons 218 (valine to leucine) and 248 (arginine to tryptophan) of p53. Bacterial cloning of p53 from SNU-C5 cells showed that the 248trp and 218leu mutants were both expressed and on separate alleles. 248trp is a common 'hot spot' mutant of p53 with variable dominant negative activity depending on the celullar context. Valine 218, in contrast, is rarely affected by mutation in cancers and is located in a region of the hydrophobic core domain away from 'hot spot' DNA contact sights. However, valine 218 is completely conserved across species, prompting us to investigate the function of 218leu in SNU-C5 cells. SNU-C5 cells exhibited complete loss of normal p53 function as evidenced by over-expression of p53 protein and by failure to show induction of p53, waf-1, mdm-2 or G1/S arrest in response to the DNA damaging agent, bleomycin. In a yeast p53 functional assay (FASAY), 50% of the clones were unable to transactivate a p53-specific promoter required for yeast colony expansion at 25, 30 or 37 degrees C. Sequencing of the p53 insert from several randomly selected wild-type and mutant yeast clones revealed that 218leu-bearing clones retained their ability to transactivate the p53-specific promoter. As expected, the 248trp-bearing clones lost this function. These data indicate that although 218leu retains normal transactivation activity on a p53 promoter in yeast at physiological temperatures, it is not capable of normal p53 function in the presence of a 248trp allele in SNU-C5 cells. It remains unclear whether the strong dominant negative activity of 248trp in SNU-C5 cells is related to the cellular context or to an unresolved abnormality of 218leu function.

Involuntary tonic spasms of a limb due to a brain stem lacunar infarction.

BACKGROUND AND PURPOSE: Although repetitive involuntary movements are a well-recognized manifestation of carotid occlusive disease, similar movements have not been reported with a lacunar infarction outside of the basal ganglia or subthalamic nucleus. We describe a man with tonic spasms associated with a lacunar infarction in the right ventral pons. CASE DESCRIPTION: Involuntary tonic spasms of a paretic limb developed acutely in a 69-year-old hypertensive man with a clinical presentation of pure motor hemiparesis. Magnetic resonance imaging demonstrated a lacunar infarction of the ventral pons. There was no evidence for carotid occlusive disease. An electroencephalogram recorded during the movements showed no abnormality. The abnormal movements responded well to treatment with oral diazepam. CONCLUSIONS: A brain stem lacunar infarction may be associated with involuntary tonic limb spasms clinically similar to those reported as paroxysmal symptoms of multiple sclerosis.

Plasmapheresis in acute episodes of fulminant CNS inflammatory demyelination.

We present six patients with acute fulminant episodes of CNS inflammatory demyelination who responded to therapeutic plasmapheresis after failing a course of high-dose methylprednisolone administered IV. Neurologic improvement occurred in three of the patients following the second plasmapheresis. Dramatic improvement in motor function (four patients) and language (two patients) began within 2 to 14 days and persisted during the 6 to 35 months (mean, 15 months) of follow-up. Results of this uncontrolled study suggest that plasmapheresis in the absence of other immunosuppressive drugs may have a role in the treatment of severe episodes of inflammatory demyelination in a select subset of MS patients.

Paracrine control of gastric epithelial cell growth in culture by transforming growth factor-alpha.

Studying primary cultures of replicating canine oxyntic mucosal cells, we found evidence for modulation of cell growth by endogenous factors. [3H]thymidine incorporation into DNA was rapid with cells cultured in medium free of serum or added growth factors, and growth rates of these cultures were markedly dependent on plating density, indicating mitogenic control by soluble endogenous growth factors. Data indicated that endogenous transforming growth factor-alpha (TGF-alpha) exerted mitogenic control under the following conditions. 1) TGF-alpha was detected in the cultured cells by radioimmunoassay and immunohistochemistry. 2) TGF-alpha-like immunoreactivity and receptor reactivity were present in the medium in concentrations sufficient to exert mitogenic control. 3) Receptors for TGF-alpha and epidermal growth factor (EGF) were present in the cultures. 4) Immunoabsorption by a TGF-alpha-specific antisera reduced [3H]thymidine incorporation. TGF-alpha was localized to parietal cells by immunohistochemistry and cell separation. In contrast, combined [3H]thymidine autoradiography and immunohistochemistry with anti-TGF-alpha did not detect TGF-alpha in dividing cells. We conclude that parietal cell TGF-alpha exerts paracrine control of mucosal cell growth in vitro, and we speculate that this is an important paracrine mechanism in vivo.

Helicobacter pylori secretes a chemotactic factor for monocytes and neutrophils.

Helicobacter pylori is associated with an inflammatory reaction in the stomach and duodenum, yet the mechanism of this inflammatory infiltrate is unknown. The ability of Helicobacter pylori to secrete a factor that attracts leucocytes is investigated. Helicobacter pylori conditioned supernatant attracted neutrophils and monocytes with 50-100% of the activity of control chemotactic factor, 10(-8) M formyl-methionine-leucine-phenylalanine. Strains derived from individuals with ulcer or non-ulcer associated H pylori infections displayed similar chemotactic activity. Preliminary characterisation shows that the factor has a molecular weight of less than 3000, is heat stable, is acid resistant, and can be diluted at least 10-fold. Checkerboard analysis confirmed that the activity was chemotactic rather than chemokinetic. This chemotactic activity could play a role in the pathogenesis of Helicobacter pylori gastritis.

Autonomous proliferation of colon cancer cells that coexpress transforming growth factor alpha and its receptor. Variable effects of receptor-blocking antibody.

Four human colon adenocarcinoma cell lines, SNU-C1, SNU-C4, SNU-C5, and NCI-H716, that are capable of proliferating autonomously in serum-free medium containing no added peptide growth factors were identified. All four cell lines show epidermal growth factor (EGF) receptors (EGFRs), express transforming growth factor alpha (TGF-alpha) messenger RNA, and release anti-TGF-alpha-immunoreactive molecules. The blocking anti-EGFR monoclonal antibody (mAb) 225 blocks autonomous proliferation of SNU-C1 and SNU-C4 cells. In both of these cell lines, the inhibitory effect of mAb 225 is reversible by the addition of EGF, TGF-alpha, or conditioned medium from any of the four cell lines. In contrast, autonomous proliferation of SNU-C5 and NCI-H716 cells is not inhibited by mAb 225 and is not affected by exogenous EGF, TGF-alpha, or conditioned medium. Together, these data confirm the previous finding that anti-EGFR antibodies can inhibit the proliferation of some carcinoma cell lines that coexpress TGF-alpha and EGFR. However, here it is shown that the mechanisms of autonomous proliferation of colon carcinoma cell lines are heterogeneous and not always sensitive to antibody disruption of TGF-alpha/EGFR autocrine interactions.

Effect of age on gastric acid secretion and serum gastrin concentrations in healthy men and women.

The effects of age on basal, meal-stimulated, and human gastrin-17-stimulated gastric acid secretion rates and serum pepsinogen concentrations were evaluated in 41 healthy men and women. Older subjects (ages 44-71 years; mean, 57 years) had higher mean basal, meal-stimulated, and gastrin-17-stimulated acid secretory rates and basal serum pepsinogen I and II concentrations than younger subjects (ages 23-42 years; mean, 33 years). Age-related differences in acid secretion were especially prominent in men, and age-related differences in serum pepsinogen I and II concentrations were more prominent in women. Higher gastric acid secretion rates in older subjects could not be explained by body size (height, weight, body surface area, or fat-free body mass) or by the higher incidence of infection with Helicobacter pylori. Using a multivariate linear regression model, age had an independent positive effect on acid secretion, and H. pylori infection had an independent negative effect. It was concluded that aging is associated with an increase in gastric acid secretion in humans, especially in men, while infection with H. pylori is associated with lower acid secretion rates.

Autocrine growth factors and solid tumor malignancy.

The ability of malignant cells to escape the constraint that normally regulate cell growth and differentiation has been a primary focus of attention for investigators of cancer cell biology. An outcome of this attention has been the discovery that the protein products of oncogenes play a role in the activation of growth signal pathways. A second outcome, possibly related to abnormal oncogene expression, has been the discovery that malignant cells frequently show an ability to regulate their own growth by the release of autocrine growth modulatory substances. Most important, the growth of certain malignant cell types has been shown to depend on autocrine growth circuits. A malignant tumor whose continued growth depends on the release of an autocrine growth factor may be vulnerable to treatment with specific receptor antagonists or immunoneutralizing antibodies designed to break the autocrine circuit. Information is rapidly emerging concerning autocrine growth factors in selected human solid tissue malignancy.

Elevation of meal-stimulated gastrin release in subjects with Helicobacter pylori infection: reversal by low intragastric pH.

The aim of the present study was to determine whether the association between Helicobacter pylori infection and increased concentrations of gastrin in serum is independent of chronic duodenal ulcer disease and whether the mechanism of this association involves a disturbance of feedback inhibition of gastrin release by intragastric acid. Of 48 subjects evaluated, 26 (54%) were seropositive for H. pylori by ELISA. Fasting and peptone meal-stimulated gastrin release at pH 2.5 and pH 5.5 as well as integrated 24-hour plasma gastrin concentrations were significantly higher in the seropositive group, even when subjects with a history of duodenal ulcer were excluded. The inhibitory effect of low pH on the release of gastrin was not attenuated in subjects with positive results in the ELISA. These data indicate that the association between seropositivity for H. pylori and enhanced release of gastrin is independent of a history of duodenal ulcer and is not caused by a disturbance of the normal feedback inhibition of gastrin release by intragastric acid.

Positive serum antibody and negative tissue staining for Helicobacter pylori in subjects with atrophic body gastritis.

Helicobacter pylori is rarely found in gastric biopsy specimens from individuals with atrophic gastritis of the body mucosa. To determine if subjects with atrophic body gastritis have evidence of previous infection with H. pylori, immunoglobulin G antibody to H. pylori was measured by enzyme-linked immunosorbent assay in sera of 399 Finnish subjects. In 124 subjects, multiple biopsy specimens from body and antrum had been evaluated for the presence of H. pylori by Giemsa staining. Antibody correlated well with H. pylori staining except in the subgroup with atrophic body gastritis, in whom the prevalence of seropositivity (86%) was significantly greater than the prevalence of positive staining (33%) (P less than 0.001). Twenty-five subjects had positive antibody and negative staining. This group had a significantly higher prevalence of atrophic body gastritis (80%), lower maximal acid output, lower serum pepsinogen I levels, and higher serum gastrin concentrations than did seropositive subjects with H. pylori. These data suggest that most patients with atrophic body gastritis, despite having a low incidence of current overt infection, have been infected with H. pylori at some point in their lives.

Prolonged inhibition of acid secretion causes hypergastrinaemia without altering pH inhibition of gastrin release in humans.

Hypergastrinaemia induced by potent inhibitors of acid secretion is thought to occur as a result of the elimination of the inhibitory effects of intragastric acid on gastrin release. The present study was designed to determine if the mechanisms responsible for feedback inhibition of gastrin release and acid secretion by intragastric acid are preserved during four weeks of varying degrees of drug-induced acid inhibition. Forty-eight healthy male volunteers were randomly assigned to one of four treatments for four weeks: 10 mg omeprazole o.m., 20 mg omeprazole o.m., 40 mg omeprazole o.m. or 150 mg ranitidine b.d. Gastrin release and acid secretion in response to peptone meals maintained at pH 2.5 and pH 5.5 by intragastric titration, and 24-hour gastrin profiles in response to standard meals were determined before treatment, at the fourth week of treatment and two weeks after discontinuing treatment. As expected, omeprazole produced dose-related effects on acid secretion and gastrin concentrations that were largely reversed after treatment was discontinued. Gastrin release in response to pH 5.5 peptone meals remained significantly greater than gastrin release in response to pH 2.5 meals during treatment with all doses of omeprazole. The ratio of pH 5.5/pH 2.5 peptone meal-stimulated gastrin release was approximately 1.5, and remained constant for all treatment groups throughout the study period. These data indicate that four weeks of drug induced hypochlorhydria causes an apparent increase in overall G-cell function, but it does not interfere with normal feedback inhibition of gastrin release and acid secretion mediated by intragastric acidity.

The gastrin hypothesis. Implications for antisecretory drug selection.

Newer potent and long-acting inhibitors of acid secretion, such as the proton pump inhibitor omeprazole, are becoming available for general use. These drugs promise to control acid-peptic disease effectively in patients who do not respond adequately to conventional short-acting H2-receptor antagonists. The safety of chronic administration of these drugs has come into question, however. Lifelong profound inhibition of acid secretion in rats induced by superpotent inhibitors of acid secretion or subtotal fundectomy is associated with the development of carcinoid tumors of enterochromaffin-like (ECL) cells in the gastric corpus. Available evidence supports a role of gastrin, which becomes chronically elevated in animals subjected to prolonged and profound hypochlorhydria. In humans, hypergastrinemic states such as Zollinger-Ellison syndrome and atrophic gastritis are associated with an increased risk of ECL-cell carcinoid tumors. Such observations have raised concern that humans may also be susceptible to carcinoid tumor formation in response to potent inhibitors of acid secretion. To date, however, no cases of carcinoid tumor have been attributed to the use of omeprazole in humans. If achlorhydric doses are not used, significant hypergastrinemia can be avoided while effectiveness of treatment is maintained. Such measures should minimize any risk of ECL-cell carcinoid tumors in humans taking potent long-term antisecretory drugs.