H2T Cells Research Articles

074 Evolution of pathologic T-cell subsets in atopic dermatitis from infancy to adulthood

The systemic immune phenotype of AD has been defined in adults and recently in young children with early disease, but chronologic changes in pediatric AD blood through adolescence have not been explored. Defining the chronological sequence of the AD phenotype is critical for leveraging new age-specific targeted treatments and prophylactic approaches. We studied immune activation and cytokine polarization in the blood of 0-5y/o, 6-11y/o, 12-17y/o and >18y/o with AD vs. age-matched controls. Using flow cytometry we measured the frequencies of activated polar CD4+/CD8+ T-cell subsets within skin-homing/CLA+vs. systemic/CLA-T-cells. Intracellular staining defined Th1/Tc1, Th2/Tc2, Th9/Tc9, Th17/Tc17, and Th22/Tc22 populations. Unsupervised clustering was used to differentiate patients based on their blood biomarker frequencies, and sharply distinguished groups aligned along a chronological spectrum. While CLA+Th1 frequencies were significantly lower in infants with AD vs. all older patients (P<0.01), CLA+Th2 T-cells were expanded similarly across all AD age groups compared with controls (P<0.05). After infancy, the CLA-Th2 frequencies were significantly increased in AD in all age groups, suggesting systemic immune activation with chronicity. While IL-22 frequencies were not increased in AD infants, they serially increased to highly significant in adolescents and adults compared to respective controls (P<0.01). Th1 frequencies nearly doubled between 0-5 and 5-12y/o in both AD (7.7% vs. 14%; P=0.03) and controls (11.9% vs. 19.5%; P=0.04), but the Th1/Th2 ratio remained significantly decreased in AD across all age groups vs. controls. Unique cytokine signatures in different patient age-groups advocate for specific rather than uniform therapeutic approaches. Comparing the profile of cleared vs persistent pediatric AD, ideally through longitudinal studies, will better define age-specific changes that predict AD clearance.

Human Adenovirus Replicates in Immunocompetent Models of Pancreatic Cancer in Syrian Hamsters

The preclinical evaluation of toxicity and antitumor effect of conditionally replicative (oncolytic) adenoviruses is hampered by the inability of human adenoviruses to replicate efficiently in murine cells. The Syrian golden hamster (Mesocricetus auratus) has been suggested as a permissive animal for adenoviral replication, and cancer cell lines derived from various hamster tumors are available. We provide evidence that wild-type adenovirus type 5 is able to infect and replicate in the pancreatic cancer cell lines HaP-T1 and H2T both in vitro and in vivo. Determination of cytopathic effect, viral spread, progeny production, and the expression of late viral proteins indicates that the complete viral cycle of adenovirus takes place, albeit less efficiently than in highly permissive human cancer cell lines A549 and HuH7. Intrahepatic inoculation of HaP-T1 and H2T cells gave rise to tumors in the liver of hamsters that resemble metastases of pancreatic cancer. The growth of HaP-T1-induced nodules was faster compared with those derived from H2T, but both caused progressive liver infiltration and peritoneal dissemination. When adenovirus was inoculated in these lesions, productive replication took place and newly formed infective virions could be recovered 4 days after administration. In conclusion, the Syrian hamster models described here offer the opportunity to evaluate the effect of oncolytic adenoviruses in an immunocompetent animal and may be a valuable tool in the preclinical evaluation of these agents.

Variable effect of streptozotocin-diabetes on the growth of hamster pancreatic cancer (H2T) in the Syrian hamster and nude mouse

Background: Streptozotocin-diabetes prevents induction of pancreatic tumors in several animal models and inhibits the growth of established human pancreatic cancer implants in nude mice. However, it also promotes growth of the hamster pancreatic cancer cell line, H2T, in the Syrian hamster. To test the hypothesis that these contradictory effects are due to tumor host differences, the growth of the H2T cell line was examined in the streptozotocin-diabetic nude mouse. Methods: H2T cells were implanted subcutaneously into streptozotocin-diabetic nude mice ( n = 10) and untreated control mice ( n = 10). After 21 days, tumors were excised and weighed. Plasma insulin and somatostatin were determined by radioimmunoassay. Results: After 3 weeks, tumors in the control group weighed 118 mg and tumors in the diabetic group weighed 28 mg ( p < 0.001). Plasma insulin was significantly decreased in the streptozotocin-treated animals compared with control animals (insulin, 23 μ U/ml vs 31 μ U/ml; p < 0.001). In contrast, somatostatin was significantly elevated in the streptozotocin-diabetic group compared with the control group (somatostatin, 179 pg/ml versus 54 pg/ml, p <0.001). Competitive binding studies revealed specific cell surface receptors for insulin (K d, 15.5 nmol/L), and somatostatin (K d, 2.5 nmol/L) on the H2T cells. In an in vitro cell proliferation assay, cell division was promoted by insulin ( p < 0.01, maximum +11%) and inhibited by somatostatin ( p < 0.01, maximum –18%). Conclusions: The variable effect of streptozotocin-diabetes on pancreatic cancer growth is due to differences in the tumor host. The growth of pancreatic cancer, particularly in streptozotocin-diabetic nude mice, may be influenced by gut peptides in a receptor-dependent fashion.(Surgery 1998;123:315-20.)

Antitumor effect of positively charged resin in the hamster cheek pouch model.

Following the signal observation that contact with positively charged dextran resin (PCDR) inhibited the growth of cultured mammary (Hs578T and MDA-MB-231), pancreatic (H2T), and myeloma (RR-658) tumor cell lines, studies were developed in the hamster cheek pouch model using hamster H2T pancreatic tumor cells to determine if the antiproliferative effect of PCDR could inhibit tumorigenesis. In these studies, the control population represented groups injected with H2T cells alone or in combination with either neutral or negatively charged resin. When cells (5 x 10(2) to 1 x 10(5)) and PCDR were administered simultaneously, the tumor incidence (percent engraftment) and growth of tumors that already had been established were significantly reduced. When PCDR was injected into already established 1-35-mm2 H2T tumors (engraftment for 21 days = 96%), the resin suppressed the growth of the smallest tumors (< 10 mm2). In none of these trials was the somatic growth of the host hamsters affected. PCDR contact with H2T cells in vitro for 4 days or used to treat growing solid tumors for 72 days significantly reduced cellular ornithine decarboxylase activity. While the mechanism of PCDR action has not been established, the observations have implications for in vivo tumor therapeutic models.

Bombesin inhibits growth of pancreatic ductal adenocarcinoma (H2T) in nude mice.

Bombesin (BBS), a tetradecapeptide, stimulates growth of various types of cells, including fibroblasts and human small cell lung cancer, and has been termed the universal "on-switch" due to its ability to stimulate the release of numerous hormones. In addition, BBS receptors have been identified in normal and neoplastic pancreatic tissue. A pancreatic ductal adenocarcinoma cell line (H2T), established in our laboratory, possesses specific binding sites for BBS. The purpose of this study was to examine the effect of BBS on the growth of H2T tumors transplanted into athymic nude mice. H2T cells (5 x 10(6) cells/mouse) were injected s.c. into the interscapular region of the nude mice and then the mice were randomized into two groups (n = 10/group). Mice received either 0.1 ml of saline with 0.1% bovine serum albumin (BSA) (control) or 0.1 ml BBS (5 micrograms/kg) intraperitoneally, three times/day. Tumor area was measured twice weekly until the mice were killed (day 32), when tumor and normal pancreas were removed, weighted, and assayed for DNA and protein content. Administration of BBS significantly inhibited H2T tumor area, weight, and DNA and protein content. Conversely, growth of normal pancreas, removed as an in vivo bioassay so as to ensure the efficacy of BBS, was stimulated. We conclude that BBS is a growth inhibitory factor for H2T tumors and that different mechanisms may be responsible for the differential growth effects elicited by normal and neoplastic pancreas in response to BBS.

Differential effect of submandibular gland resection on the growth of pancreatic cancer in cheek pouch and subcutaneous tissue

Exogenous administration of epidermal growth factor (EGF) enhances tumor growth of H2T, a hamster pancreatic cancer that is inoculated in the cheek pouch. The effect of endogenous EGF on tumor growth, however, is not known. The main source of EGF in the body is the submandibular glands. This study examined the influence of submandibular gland resection (SMx) on H2T tumor growth in the cheek pouch and compared it to the growth in SC tissue. Bilateral SMx or sham operation was performed on male Syrian golden hamsters. In 30 hamsters (n = 15 for each operation group), 5 x 10(4) and 5 x 10(5) H2T cells were inoculated in the bilateral cheek pouches and intrascapular SC tissue, respectively (Exp. 1). In another 30 hamsters (n = 15 for each operation group), 5 x 10(5) and 1 x 10(6) H2T cells were inoculated at the same sites (Exp. 2). Two longest perpendicular diameters of the tumor were measured once a week for 12 wk (Exp. 1) or 8 wk (Exp. 2), and the tumor area was calculated. The tumor area in the cheek pouch became significantly smaller in the SMx group than the sham-operated group after the 8th wk (Exp. 1) or the 7th wk (Exp. 2). On the other hand, the tumor area in the SC tissue did not show any difference between groups through the experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Cortivazol increases glucocorticoid receptor expression and inhibits growth of hamster pancreatic cancer (H2T) in vivo.

Glucocorticoids are effective in the treatment of certain leukemias and lymphomas, but their effects on the growth of several solid tumors have not been determined. We report here that cortivazol (CVZ), a potent synthetic glucocorticoid, inhibits the growth of a hamster pancreatic adenocarcinoma, H2T, in vivo. CVZ regulation of glucocorticoid receptor (GR) expression was followed as a specific molecular correlate. H2T cells were injected into cheek pouches of male Syrian golden hamsters, where they formed readily measurable tumors. Two studies were performed. In the first, hamsters were randomized to three groups immediately after injection of tumor cells: control, CVZ (0.1 micrograms/g body wt), or CVZ (0.3 micrograms/g body wt). Injections of either CVZ or its vehicle were administered on a 14-day cycle of 5 treatment days, followed by 9 days off treatment. Tumors were measured and areas calculated weekly. On day 48, the hamsters were killed and the tumors excised, weighed, and analyzed for DNA, RNA, and protein content. In the second study, randomization and treatment schedule were as before, except that on day 33 the hamsters were killed, tumors were excised and weighed, and total RNA from the tumors was isolated. GR mRNA content was determined by filter hybridization with a 32P-labeled GR cDNA probe, and the signal normalized by reprobing for alpha-tubulin as an invariant, independent signal. At either dose, CVZ significantly inhibited H2T tumor area and weight and DNA, RNA, and protein content. Body weights of animals treated with CVZ were not significantly decreased as compared with controls. In addition, GR mRNA in H2T cells was increased approximately twofold by CVZ.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of pancreatic adenocarcinoma cell growth by lovastatin

RAS protein (p21 ras) requires farnesyl (an intermediate of cholesterol synthesis) for activation. Activating mutations of K-ras gene have been detected in most human pancreatic adenocarcinomas. In the present study, the effect of lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A, the rate-limiting enzyme of cholesterol synthesis, on the growth of five pancreatic cancer cell lines (human-CAV, MIA Paca2, CAPAN2 and PANC1, and hamster-H2T) in vitro and of two cell lines (CAV and H2T) in vivo was examined. Inhibition of cell growth was observed with lovastatin doses at or above 2.5 μg/mL for H2T, CAV, MIA Paca2, and CAPAN2 or 10 μg/mL in PANC1. The H2T cell line was studied further to determine the reversibility of growth inhibition. Mevalonic acid (1 mmol/L) reversed lovastatin-induced inhibition of cell growth if it was added with lovastatin (2.5 μg/mL). Similarly, removal of lovastatin from the medium within 24 hours after treatment allowed recovery of cell growth. The effect of lovastatin on cell growth was irreversible after 48 hours of exposure. The survival fraction of H2T cells was markedly decreased by 1- or 24-hour exposure to 75 μg/mL but not to doses ranging from 0.5 to 60 μg/mL of lovastatin. Growth of pancreatic carcinoma xenografts (CAV and H2T) in nude mice was inhibited by a subcutaneous infusion of lovastatin (50 μg/h). These results indicate that mevalonic acid or a metabolite in the cholesterol synthesis pathway is necessary for growth of pancreatic cancer cells and suggest that lovastatin should be further examined as a potential therapeutic agent for pancreatic cancer.

Further studies of enhanced growth of pancreatic carcinoma in diabetes

Clinical studies suggest that pancreatic cancer occurs more often in persons with diabetes mellitus [1–7]. We have previously shown that the hamster pancreatic carcinoma cell line H2T grows more rapidly when implanted in streptozotocin (STZ)-diabetic hamsters [8]. To determine if enhanced growth of pancreatic carcinoma cells in diabetic hamsters is due to polyphagia associated with diabetes, H2T cells were implanted into the cheek pouch of three groups of animals: normal hamsters (group I), STZ-diabetic hamsters (group II), and STZ-diabetics pairfed to normals (group III). Tumor weights 30 days after implantation were 172 g in group I, 368 g in group II, and 369 g in group III ( P < 0.007). There was no significant difference between the two diabetic groups. Thus, STZ diabetes appears to promote the growth of pancreatic carcinoma cells by a mechanism other than increased nutrient intake by diabetic tumor hosts.

Neuropeptide Y and peptide YY stimulate the growth of exocrine pancreatic carcinoma cells

Neuropeptides exert inhibitory effects on pancreatic secretion, but their role in the regulation of growth is unknown. This study was executed to evaluate the effects of PYY and NPY on cell growth and 3H-thymidine incorporation in human (MiaPaCa-2, Capan-2) and hamster (H2T) exocrine pancreatic carcinoma cells in vitro. A significant increase in the number of cells after 96 h of treatment with NPY was observed at 0.01 μM in H2T, 0.1 μM in MiaPaCa-2 and at 1 μM in Capan-2 cells. PYY was less potent and did not increase significantly cell growth in MiaPaCa-2, but did at 0.1 μM in Capan-2 and at 1 μM concentration in H2T. Stimulation for 48 h with NPY increased 3H-thymidine incorporation significantly at 0.01 μM in all cell lines. With PYY, stimulation of 3H-thymidine incorporation occurred in H2T cells at 0.01 μM. 3H-thymidine incorporation after PYY treatment was significantly increased at 0.1 μM in MiaPaCa-2 and at 1 μM in Capan-2 cells. Receptor studies showed low but definite specific binding of both NPY and PYY in all cell lines. The results suggest that NPY and PYY may have a role in the regulation of growth of exocrine pancreatic carcinoma cells.

The Effects of 2-Deoxy-D-Glucoseand α-Difluoromethylornithine on the Growth of Pancreatic Cancer in Vivo

The glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), inhibits growth of some cancers. alpha-Difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis. We and others have previously shown that DFMO inhibits cancer growth in a number of models. The present study was designed to investigate the effects of 2-DG alone and combined with DFMO on the growth of H2T hamster pancreatic ductal adenocarcinoma. Twenty-eight male Syrian golden hamsters were inoculated with 500,000 H2T cells, and then randomized into four groups of seven each: group 1 served as control; group 2 received DFMO (3% in drinking water); group 3 received 2-DG (500 mg/kg/day) intraperitoneally; group 4 received a combination of 2-DG and DFMO. Treatment began 5 days after tumor cell inoculation and continued for 28 days. At the end of the treatment period, the area of the H2T tumor was reduced 31% by DFMO compared with a 22% reduction caused by 2-DG. Tumor weight was significantly reduced (31%) by DFMO but not by 2-DG. Tumor contents of DNA, RNA, and protein were also reduced by DFMO but not 2-DG. Tumor concentration of the polyamines, putrescine and spermidine, were reduced by DFMO, but 2-DG did not alter levels of polyamines. The combination of DFMO and 2-DG caused a significantly greater reduction in tumor weight and putrescine content compared with DFMO alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclosporine and alpha-difluoromethylornithine exhibit differential effects on colon and pancreatic cancer in vitro.

alpha-Difluoromethylornithine (DFMO) is a known irreversible inhibitor of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. Cyclosporine (CsA) has been reported to inhibit ODC activity in vitro. In the present study, we compared the effects of DFMO and CsA on growth, survival, and polyamine levels in mouse colon cancer (MC-26) and hamster pancreatic cancer (H2T) cells in vitro. The growth and survival of MC-26 and H2T cells were inhibited by both DFMO and CsA. However, H2T cells were observed to be significantly more sensitive than MC-26 cells to both CsA and DFMO. The inhibitory effects of CsA were blocked by the addition of the polyamine, putrescine, in both MC-26 and H2T cells. Polyamine levels were altered significantly in both MC-26 and H2T cells treated with CsA and DFMO. However, the profile of these alterations differed between MC-26 and H2T cell lines. Putrescine and spermidine levels in MC-26 cells were more sensitive to DFMO inhibition than were H2T cells. Spermine levels were consistently elevated in MC-26 cells exposed to CsA or DFMO, while the level of spermine in H2T cells decreased significantly in response to the same drugs. These results suggest that CsA and DFMO exhibit different effects on colon and pancreatic cancer growth in vitro. In addition, the differences in the sensitivity of pancreatic and colon cancer to CsA and DFMO indicate potentially important differences in polyamine metabolism between the two cell lines.

Treatment of Hamster Pancreatic Cancer With α-Difluoromethylornithine, an Inhibitor of Polyamine Biosynthesis&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="FN3"&gt;3&lt;/xref&gt;

Polyamines are essential for cell division and growth. Inhibition of polyamine biosynthesis by alpha-difluoromethylornithine (DFMO) on the growth of hamster H2T pancreatic cancer was investigated both in vitro and in vivo. Cell-doubling time (TD) and survival fraction were determined after a single treatment with DFMO (5 mM). We examined the ability of putrescine to reverse the growth-inhibitory effect of DFMO. The TD for cells treated with DFMO in vitro was 49.6 +/- 5.7 versus 25.4 +/- 2.6 hours for control. The addition of putrescine to DFMO-treated H2T cells showed a reversal of the growth-inhibitory effect of DFMO. Cytotoxicity in vitro increased with prolonged treatment; the survival fraction after 24 hours of treatment was 32%; after 48 hours, 19%; after 72 hours, 13%; and after 92 hours, 8%. We performed two separate animal experiments. In experiment I, H2T cells were injected into the cheek pouch of male Syrian golden hamsters; controls did not receive DFMO. Continuous treatment with 3% DFMO in the drinking water was begun 7 days before, on the day of, or 7 days after tumor cell injection. In experiment II, 4 groups were treated identically to those in experiment I. An additional group of 10 hamsters received 3% DFMO and no tumor, and another additional group of 10 hamsters were housed individually with 3% DFMO begun 7 days after tumor cell injection. Tumor size, body weight, water, and food intake were measured. DFMO treatment in vivo significantly inhibited tumor size and inhibited growth of pancreatic cancer by as much as 50% of control. Our results demonstrate a significant antiproliferative effect of DFMO on the growth of pancreatic adenocarcinoma both in vitro and in vivo.