Global analysis of the human gastric epithelial transcriptome altered by Helicobacter pylori eradication in vivo.

OBJECTIVE: The transcriptional profile of gastric epithelial cell lines cocultured with Helicobacter pylori and the global gene expression of whole gastric mucosa has been described previously. We aimed to overcome limitations of previous studies by determining the effects of H pylori eradication on the transcriptome of purified human gastric epithelium using each patient as their own control. DESIGN: Laser capture microdissection (LCM) was used to extract mRNA from paraffin-embedded antral epithelium from 10 patients with peptic ulcer disease, before and after H pylori eradication. mRNA was reverse transcribed and applied on to Affymetrix cDNA microarray chips customised for formalin-fixed tissue. Differentially expressed genes were identified and a subset validated by real-time polymerase chain reaction (PCR). RESULTS: A total of 13 817 transcripts decreased and 9680 increased after H pylori eradication. Applying cut-off criteria (p<0.02, fold-change threshold 2.5) reduced the sample to 98 differentially expressed genes. Genes detected included those previously implicated in H pylori pathophysiology such as interleukin 8, chemokine ligand 3, beta defensin and somatostatin, as well as novel genes such as GDDR (TFIZ1), chemokine receptors 7 and 8, and gastrokine. CONCLUSIONS: LCM of archival specimens has enabled the identification of gastric epithelial genes whose expression is considerably altered after H pylori eradication. This study has confirmed the presence of genes previously implicated in the pathogenesis of H pylori, as well as highlighted novel candidates for further investigation.

Multidrug resistance-1 and p-glycoprotein in human chondrosarcoma cell lines: expression correlates with decreased intracellular doxorubicin and in vitro chemoresistance.

We report on two chondrosarcoma cell lines, FS and AQ, that may be used as models of multidrug resistance in chondrosarcoma. Multidrug resistance-1 expression was assayed with reverse transcription-polymerase chain reaction. Immunostaining for the multidrug resistance-1 product, P-glycoprotein, was performed with the monoclonal antibody C494. Intracellular levels of doxorubicin were measured by fluorescent emission at 590 nm after 1 hour of incubation with the agent and again after 1, 2, and 4-hour washout periods. Chemosensitivity was assayed by staining micropellet cultures of AQ and FS cells with fluorescein acetate before and after the cells were exposed to varying doses of doxorubicin for 48 hours. Cytotoxicity was assessed by comparison of computer-processed images before and after treatment. The FS cell line was positive for multidrug resistance-1 expression, stained heavily for P-glycoprotein, and had significantly lower intracellular levels of doxorubicin than the AQ cell line, which was negative for multidrug resistance-1 and P-glycoprotein. Chemosensitivity testing showed that the FS cell line was significantly more resistant to doxorubicin than was the AQ cell line at all doses tested. Our results show that multidrug resistance-1 expression in a human chondrosarcoma cell line results in resistance to doxorubicin in vitro.

Unexpected in vitro chemosensitivity of malignant gliomas to 4-hydroxyperoxycyclophosphamide (4-HC).

To individually tailor chemotherapy for patients with malignant gliomas according to tumor chemosensitivity, a rapid assay system which can be performed with a high success rate is needed. The fluorescent cytoprint assay (FCA) can assess multiple chemotherapeutic agents using small (approximately 500 cells) tumor aggregates very quickly (approximately 1 wk). Tissue samples from 51 patients with malignant gliomas obtained either at time of initial diagnosis (n = 34) or at recurrence were assayed using this method. The assay success rate approached 90% in those culture samples which were histologically verified as tumor. A meaningful number of agents could be tested both on samples obtained by stereotactic biopsy (median, 5) and on specimens from more extensive resections (median, 6). One hundred ninety-three FCAs were performed on a samples obtained from 36 patients. In only twenty six assays (14%) was an agent deemed sensitive (> 90% cell kill) to a chemotherapeutic agent. Sixty-two percent of sensitive FCAs were observed in tumors tested against the activated analog of cyclophosphamide, 4-hydroxyperoxycyclophosphamide (4-HC), where a sensitivity rate (# samples sensitive/total tested against agent) of 64% (95 % CI, 36.6-77.9%) was noted. This rate was significantly higher than with any other agent tested (p = 0.012, two sided McNemar's test) and was not affected by age, histology or disease status. We conclude that: (1) the FCA represents a feasible method for quickly assaying tumors for sensitivity to multiple chemotherapeutic agents; and (ii) malignant gliomas may be particularly sensitive to 4-HC.

In vivo selection of a highly metastatic cell line from a human pancreatic carcinoma in the nude mouse.

A cell line with high metastatic capacity to the liver was established by sequential passages of a human pancreatic cancer cell line through the nude mouse liver. A subline, L3.5, established after five passages of the fast-growing variant (FG) of the human pancreatic cancer COLO 357 through the nude mouse liver produced extensive hepatic metastases in 100% of experimental animals when injected into the spleen. The incidence of pulmonary metastases decreased from 43% for FG to 9% for L3.5. The L3.5 cell line showed aggressive growth with almost complete replacement of the hepatic parenchyma in one third of the mean time required for the development of macroscopic metastases of FG in the liver after splenic injections of tumor cells. This study indicates that the nude mouse provides a good model for in vivo selection of metastatic cells from human pancreatic cancer. The L3.5 cell line will be valuable in the study of human pancreatic cancer metastasis, particularly in the area of survival and growth of metastatic cells in the microenvironment of the liver.

The fluorescent cytoprint assay: a new approach to in vitro chemosensitivity testing.

Today's chemotherapy is protocol driven and based on regimens derived from extensive clinical trials. Often, several proven regimens are available for a particular cancer, and little more than cost restrictions direct the physician's choice of protocol. Unfortunately, all patients do not respond to all regimens. Initial effective therapy is essential for good cancer control and patient management. Therefore, a predictive in vitro chemosensitivity test would be valuable in selecting treatment for individual patients. The fluorescent cytoprint assay (FCA) is an in vitro chemosensitivity assay that tests tumor specimens maintained in microorgan culture. The FCA has an assay success rate of 94% or better for most solid tumor types and predictive accuracies of 86% and 91% for positive and negative clinical responses, respectively. This paper summarizes 6 years of experience with this assay, discusses its advantages as a reliable predictive assay, and defends its place in chemotherapy decisions for individual patients.

Predictive value of the fluorescent cytoprint assay (FCA): a retrospective correlation study of in vitro chemosensitivity and individual responses to chemotherapy.

The potential clinical usefulness of the fluorescent cytoprint assay (FCA) was assessed retrospectively in 73 cancer patients by correlating individual tumor chemosensitivity in vitro with responses to chemotherapy. The data show that the FCA has a sensitivity of 98%, specificity of 81%, and predictive accuracies of 85% and 97% for positive and negative clinical responses, respectively.

An analysis of immunocomplexes for the detection of the early stages of colon cancer.

A radioimmunoassay was developed for the detection of the early stages of colon cancer by analysis of immune complexes (IC) with a specific polyclonal antibody. Human colon cancer cells were grown in a capillary culture system to provide unaltered antigens for the development of a specific antibody. The antibody was labeled with iodine 125 (125I) and used to analyze the antigen component of IC removed from whole serum. The assay was positive in 50% and 88% of known Dukes' A and Dukes' B colon cancer patients, respectively. It was also positive for only 25% of Dukes' C and 14% of Dukes' D patients, possibly because of the decreased quantity of specific IC found in the late stages of colon cancer. A blind study of patients referred for colonoscopy compared pathology diagnosis with the test results. The assay was positive for one patient with a polypoid adenocarcinoma (Dukes' B) and one with a villous adenoma and negative for 38 patients with benign polyps and 43 with no polyps. The assay was negative for all patients with stomach cancer and inflammatory bowel diseases and positive for about 10% of the patients with pancreas or breast cancer. The results of this preliminary investigation suggest that this radioimmunoassay may be useful for the detection of the early stages of colon cancer.

Heterogeneity of potential for hematogenous metastasis in a human pancreatic carcinoma.

Human pancreatic carcinoma, a disease with grave prognosis, frequently metastasizes to the liver, with detrimental consequences for the host. Good models of experimental metastasis for this disease are lacking. We describe a model of hepatic metastasis from the fast-growing variant (FG) of the human pancreatic carcinoma COLO 357. We also show that the slow-growing variant (SG) of COLO 357 lacks the potential for forming hepatic and pulmonary metastases following injection into the spleen of the nude mouse. This expression of heterogeneity of potential for hematogenous metastases can be exploited by pursuing studies aiming at identifying differences between the cells with and without metastatic potential.

Cellular viability in human tumor micro-organ cultures: in situ quantitation by image processing.

At present, cytotoxicity measurements using the fluorescent cytoprint assay are based on achieving complete cell death in cultures of drug-sensitive tumors. Thus, the usefulness of the assay would be extended if partial effects of chemotherapeutic drugs could be quantified. In this study, we addressed the issue by developing and validating a thresholding algorithm for automatic image processing that can be used to quantify the areas occupied by viable (i.e., fluorescent) micro-organs in the culture.

"COLO 357," a human pancreatic adenosquamous carcinoma: growth in artificial capillary culture and in nude mice.

The human pancreatic cancer cell line COLO 357 has been xenografted s.c. in athymic Swiss mice. The xenografts grew well to form typical adenosquamous carcinomas. The cells were placed in a perfused artificial capillary system where they formed a solid tumor mass which survived for 7 weeks. In this system, the cells consumed glucose and released enzymes and carcinoembryonic antigen into the extracapillary space.

Establishment and characterization of two human pancreatic cancer cell lines tumorigenic in athymic mice.

Two human pancreatic cancer lines, RWP-1 and RWP-2, have been established from 2 patients with primary pancreatic cancer metastatic to the liver. The patients' tumors, the xenografted tumors, and tumors obtained by inoculation of nude mice with cultured RWP-1 and RWP-2 cells are all moderately-well-differentiated ductal cell adenocarcinomas. Ultrastructural analysis supports the tissue histopathology findings. Xenografts of RWP-1 tumors double every 10 days, whereas the doubling time of RWP-2 xenografts is 22 days. Both tumors contain mucin. RWP-1 and RWP-2 cells have a doubling time in culture of 45 hr and form colonies in soft agar. RWP-1 cultures appear to be morphologically heterogeneous; two distinct epithelial cell types can be identified. RWP-1 and RWP-2 have modal chromosome numbers of 64 and 62, respectively. Appreciable levels of glucose-6-phosphate dehydrogenase and lactic dehydrogenase were found in both cell lines and xenografts. RWP-1 and RWP-2 cells produce appreciable amounts of carcinoembryonic antigen, 1090 and 414 ng/10(6) cells, respectively.

Bovine pepsinogens and pepsins. The sequence around a reactive aspartyl residue.

The specific inhibitor, N-diazoacetylnorleucine methyl ester reacts stoicheiometrically with bovine pepsin resulting in a simultaneous loss of all enzymic activity. A peptide containing a modified aspartyl group was isolated from bovine pepsin labelled with (14)C-labelled inhibitor. The aspartic acid residue is presumed to be part of the active centre and is in the same heptapeptide sequence as in porcine pepsin: Ile-Val-Asp-Thr-Gly-Thr-Ser.

Bovine pepsinogens and pepsins. A series of zymogens and enzymes that differ in organic phosphate content.

Several minor pepsinogens, present in extracts of bovine fundic mucosa obtained from the fourth stomach or abomasum, were separated from the main pepsinogen by chromatography on hydroxyapatite at pH7.3. The major pepsinogen and two of these minor pepsinogens were studied in detail. All three zymogens have N-terminal Ser-Val-, C-terminal -Val-Ala and not more than 1mol of glucose/mol of protein; no significant differences in amino acid composition were found. The pepsinogens differ in their organic phosphate content, which accounts for their chromatographic separation. By activation at 0 degrees C and pH2, a corresponding series of pepsins is formed. These enzymes were separated by hydroxyapatite chromatography at pH5.7. All the pepsins have N-terminal valine, C-terminal alanine and are free from carbohydrate. Again the only difference detected among them is in their organic phosphate content. The pepsins of high phosphate content are converted by an acid phosphatase in vitro into pepsins of low phosphate content.