Colon cancer cells cultured under hyperosmotic conditions as in vitro model to investigate dehydration effects on cancer drug susceptibility.

BACKGROUND: In most clinical studies older people are underrepresented compared to the demographic reality. However, risk for some severe diseases like cancer typically increase with age. Most insight into cancer treatment comes from mixed-age patient cohorts, leading to a lack of detailed understanding of cancer drug effects in the elderly population. There is growing evidence that cancer drug effects can be influenced by dehydration conditions often found in older people. Colon cancer remains the second leading cause of death by cancer in Europe. Inter- and intra-heterogeneity of tumors contribute to why some individuals do not respond to specific cancer therapies or may often suffer a relapse. OBJECTIVE: Our study applies an in vitro drug test system for simulating treatment with cytostatics of colorectal cancer in elderly patients with dehydration condition. METHODS: Two well-known colon cancer cell lines, Caco-2 and RKO, harboring defined cancer-related mutations, were step-wisely adapted from routine culture medium to a severe hyperosmotic condition (397 mOmol/kg) by adding sodium chloride to the medium. We investigated the effects of these cell culture conditions, which should mimic cellular dehydration in elderly people, on the growth characteristics of the cells. Therefore, cell proliferation was investigated by measuring population doubling times. Furthermore, we investigated how the metabolic activity of the cells was influenced by treatment with different concentrations of cyclophosphamide (CPA) under normal and hyperosmotic conditions. RESULTS: We found that Caco-2 and RKO cell lines have an identical cell doubling time of 23 hours in normosmotic medium. However, hyperosmotic medium lifted the doubling time of Caco-2 cells to 31 hours while that of RKO cells did not change. Despite reduced cell proliferation rates, hyperosmotic medium sensitized Caco-2 cells to treatment with 10 mM CPA for 48 hours as measured by metabolic activity assays on ATP levels. CONCLUSIONS: The two investigated colon cancer cells lines reacted differently to hyperosmotic conditions. Only the growth of Caco-2 cells was reduced by increased osmolality. Despite this reduced growth their sensitivity to an alkylating cytostatic agent was even slightly increased. We are now in line to examine these effects in more detail and with more tumor cell lines.

[Endoscopic submucosal excavation (ESE) is a safe and useful technique for endoscopic removal of submucosal tumors of the stomach and the esophagus in selected cases]. / Die Endoskopische Submukosaexkavation (ESE) ist für ausgesuchte Fälle eine sichere und praktikable Technik zur endoskopischen Entfernung submuköser Tumoren von Magen und Speiseröhre.

Submucosal tumors of stomach and esophagus are often detected incidentally during endoscopy and further characterized by endoscopic ultrasonography. After risk estimation such submucosal tumors are either controlled by watchful waiting or surgically resected. Nevertheless, symptomatic submucosal tumors should be treated. Endoscopic submucosal excavation (ESE) and submucosal tunneling endoscopic resection (STER) may represent an alternative non-surgical therapeutic option. Two cases of complete endoscopic resection of symptomatic submucosal tumors are reported: a small gastrointestinal stroma tumor (GIST) of the antrum and a 12  cm long esophageal lipoma. For selected cases, ESE of symptomatic submucosal tumors of stomach and esophagus represents a useful alternative compared to surgical removal particularly if mass is located in antrum or corpus, sized < 20  mm and clearly defined by endoscopic ultrasonography.

Cholestatic liver diseases from child to adult: the diversity of MDR3 disease.

The phospholipidfloppase MDR3 (gene symbol: ABCB4) is expressed in the canalicular membrane of hepatocytes and mediates the biliary excretion of phosphatidylcholine, which is required for the formation of mixed micelles in bile. Several mutations of ABCB4 have been identified, which cause cholestatic liver diseases of varying severity including progressive familial intrahepatic cholestasis type 3 (PFIC-3), intrahepatic cholestasis of pregnancy (ICP) and the low phospholipid associated cholelithiasis syndrome (LPAC). Here, we report on four new (S1076N; L 23Hfs16X; c.286 + 1G > A; Q 1181E) and one known (S27G) MDR3 mutations in eight patients of three families. The patients presented with a wide spectrum of liver diseases. The clinical presentation and decisive laboratory findings or the association to a trend-setting family history led to the identification of the genetic background in these patients. Even the same mutation may be associated with varying disease progression.

Ceruloplasmin expression in rat liver cells is attenuated by insulin: role of FoxO transcription factors.

The phosphoinositide 3'-kinase (PI3 K)/Akt pathway controls the activity of a number of proteins important in the regulation of apoptosis and cell proliferation. FoxO (forkhead box, class O) transcription factors, substrates of the Ser/Thr kinase Akt, control the expression of several target genes that are crucial to the defense against oxidative stress, the regulation of cell cycle, and apoptosis in mammalian cells. Here, expression of ceruloplasmin (CP), the major copper-containing protein in blood released by the liver, was investigated. We observed a significant downregulation of CP mRNA levels after insulin treatment in H4IIE rat hepatoma cells. The PI3K inhibitor wortmannin counteracted this insulin effect on CP mRNA levels, indicating that the PI3K/Akt cascade is involved in the regulation of CP expression. Stimulation of FoxO1 was induced in H4IIE rat hepatoma cells expressing a conditionally active FoxO1 construct, resulting in significant upregulation of CP mRNA levels. This upregulation was prevented in the presence of insulin. In parallel, mRNAs of established FoxO target genes were analyzed: like CP mRNA, selenoprotein P and glucose 6-phosphatase mRNAs were upregulated by FoxO1, which was prevented by insulin. The same effects of insulin on CP mRNA levels were detected in primary rat hepatocytes. Furthermore, CP release into cell culture media was analyzed with primary hepatocytes and found to be attenuated by insulin. In line with its insulin-mimetic effects on cultured cells, Cu (2+) imitated the effect of insulin on CP expression and caused a downregulation of CP mRNA levels in rat hepatoma cells.

Hyperosmotic activation of the CD95 death receptor system.

Apoptosis is characterized by cell shrinkage, nuclear condensation, DNA fragmentation and apoptotic body formation. These features distinguish apoptosis from other types of cell death, such as necrosis. Whereas some signs of apoptosis, such as externalization of phosphatidylserine, altered mitochondrial function or activation of caspases are cell type- and death signal-dependent, apoptotic cell volume decrease (AVD) is an early and ubiquitous event and little is known about the signalling events, which are localized upstream of the plasma membrane transport steps leading to AVD and the proapoptotic events, which are induced by osmolyte loss and cell shrinkage. In hepatocytes hyperosmotic shrinkage sensitizes the cells towards CD95 ligand-induced apoptosis by activating the CD95 system. This complex process with a NADPH oxidase-derived reactive oxygen species signal as an important upstream event, allows via Yes, JNK and epidermal growth factor-receptor activation for CD95 tyrosine phosphorylation as a prerequisite for CD95 targeting to the plasma membrane and formation of the death inducing signalling complex. Other covalent modifications such as CD95-tyrosine-nitration or CD95-serine/threonine-phosphorylation can interfere with the CD95 activation process. The findings not only provide a mechanistic explanation for the high susceptibility of dehydrated cells for apoptosis, but also give insight into the role of AVD.

The hepatocyte integrin system and cell volume sensing.

Alterations of cell volume induced by either aniso-osmotic environments or under the influence of hormones, concentrative amino acid uptake and oxidative stress were recognized as an independent signal contributing to the regulation of metabolism and gene expression. The regulation of cell function by hydration changes requires structures, which register fluctuations of cell hydration (osmosensing) and thereby activate intracellular signalling pathways towards effector sites (osmosignalling). Meanwhile, it is well established that osmosensing and signalling integrate into the overall context of hormone- and nutrient-induced signal transduction. Recent evidence suggests integrins to play a major role in osmosensing and signalling due to hepatocyte swelling. This review focuses on the role of integrins in sensing of hepatocyte swelling as triggered by hypo-osmolarity, glutamine and insulin and the relevance of integrin-dependent osmosignalling for inhibition of autophagic proteolysis, stimulation of canalicular bile acid excretion and regulatory volume decrease.

In vitro induction of tumor necrosis factor-α by ochratoxin A (OTA) from rat liver: role of Kupffer cells.

Tumor necrosis factor-α (TNF-α) is released from blood-free perfused rat liver by the fungal metabolite ochratoxin A. Here we have identified Kupffer cells as the sole source of OTA-mediated cytokine release. If single cell preparation of Kupffer cells, hepatocytes, or sinusoidal endothelial cells were prepared from rat livers, only Kupffer cells released TNF-α upon incubation with 2.5 µmol/l OTA. OTA failed to induce TNF-α release in the blood-free perfused isolated rat liver when Kupffer cells were blockedin vitro by 15 µmol/l gadolinium chloride. When rats were pretreatedin vivo with the Kupffer cell depleting clodronate liposomes, OTA-mediated TNF-α release was abrogated in the isolated perfused liver model.

Release of osmolytes from perfused rat liver on perivascular nerve stimulation: alpha-adrenergic control of osmolyte efflux from parenchymal and nonparenchymal liver cells.

The effects of perivascular nerve stimulation and phenylephrine on osmolyte release were studied in the intact perfused rat liver and isolated liver parenchymal cells (PC) and nonparenchymal cells. In the perfused liver, electrical stimulation of perivascular nerves (20 Hz/2 ms/20 V) led to a phentolamine-sensitive increase of cell hydration by 6.5% +/- 1.2% (n = 3) and a transient phentolamine-sensitive stimulation of taurine and inositol, but not betaine, release. These nerve effects were mimicked by phenylephrine, but not prostaglandin F2alpha, and were not affected by sodium nitroprusside (SNP) or ibuprofen. Nerve stimulation-induced taurine, but not inositol, release was inhibited by 4, 4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS) (50 micromol/L). Single-cell fluorescence studies with isolated liver PC, Kupffer cells (KC), sinusoidal endothelial cells (SEC), and hepatic stellate cells (HSC) revealed that phenylephrine induced an increase in cytosolic free Ca2+ only in PC and HSC, but not in KC and SEC, whereas extracellular uridine triphosphate (UTP) produced Ca2+ transients/oscillations in all liver cell types studied. Phenylephrine had no effect on osmolyte release from isolated KC and SEC, but increased taurine (but not inositol) release from PC and inositol (but not taurine) efflux from HSC. The data suggest that: 1) liver cell hydration and-consecutively-osmolyte content are modulated by hepatic nerves via an alpha-adrenergic mechanism, which does not involve eicosanoids or hemodynamic changes; 2) that PC and HSC are the primary targets for nerve-dependent alpha-adrenergic activation, whereas 3) KC and SEC probably do not express alpha-adrenoceptors coupled to Ca2+ mobilization or osmolyte efflux.

Activation of rat hepatic stellate cells in culture is associated with increased sensitivity to endothelin 1.

The effect of endothelin (ET) 1 on intracellular Ca2+ transients in cultured rat hepatic stellate cells (HSCs) during transformation was studied by use of single-cell fluorescence. Regardless of the duration of HSC culture, ET-1 caused a BQ-123-sensitive but IRL-1038-insensitive elevation of [Ca2+]i, indicating the involvement of ETA but not ETB receptors. HSCs in early culture ("quiescent HSCs") were mildly responsive to ET-1: the ET-1 concentration required to obtain a [Ca2+]i transient in 50% of the cells (RC50) was 7 nmol/L, and all cells responded to ET-1 concentrations above 40 nmol/L. With culture time, -smooth muscle actin (-SMA) expression increased, as did the ET-1 sensitivity of cells, resulting in a shift of the RC50 value from 7 nmol/L to 13 pmol/L within 8 days. Independent of the duration of culture, ET-1 sensitivity was higher in -SMA-expressing cells. On the other hand, sensitivity of HSCs to produce a [Ca2+]i response to extracellular uridin 5'-triphosphate (UTP) or phenylephrine did not change during the activation process. There was no difference between quiescent and activated HSCs with respect to the sharing of intracellular Ca2+ stores, which could be mobilized by ET-1, UTP, and phenylephrine, respectively. The data suggest three conclusions. (1) A marked increase in ET-1 sensitivity of HSCs during the activation process suggests a potentiation of autocrine/paracrine stimulation. (2) HSCs are susceptible to -adrenergic and purinergic stimulation, but sensitivity to phenylephrine and UTP is not affected during the transformation process. (3) The ET-1-mobilizable Ca2+ store is contained in and is smaller than the Ca2+ pool, which is mobilized by phenylephrine or UTP.

Holtzman Inkblot Technique scores of delinquent adolescents: a replication and extension.

The Holtzman Inkblot Technique (HIT) was administered to 83 male juvenile delinquents, ranging in age from 13 to 16 years (mean age: 15.2 years), who were being detained at the Texas Youth Council Reception Center. Scores from 22 HIT variables scored were factor-analyzed and then compared with Megargee's pioneering normative HIT study of juvenile delinquents published in 1965. Results of the two investigations were strikingly similar, reinforcing Megargee's finding that norms for nondelinquent adolescents are not appropriate for use with a confined delinquent group. Important differences were discovered, however, and these are discussed.

Performance of forensic patients on the Holtzman Inkblot Technique: a normative study.

The Holtzman Inkblot Technique was administered to all 269 adult males admitted as forensic patients in the state of Texas during a 12-mo. period. Normative data presented suggest that these forensic patients differ from other criterion groups in their responses to this psychometrically rigorous projective technique. Factor analysis of the scores indicated a factorial structure similar to that reported for several of the Holtzman standardization samples but with at least one factor which seems to be unique to these forensic patients.

The Holtzman Inkblot Technique is not the Rorschach: a reply to Lockwood, Roll, and Mathews.

A previous article by Lockwood, Roll, and Matthews (1981) reported dramatic differences between the Movement scores obtained by 6-year-old children on the Holtzman Inkblot Technique (HIT) and previously reported normative data for this variable. The Lockwood et al. data were rescored by experienced HIT scorers; their findings-and other possible reasons for the high HIT Movement scores reported by Lockwood et al.-are described and discussed. Finally, the practice of using the HIT in the manner of the Rorschach is criticized.