Reactive oxygen species and redox-induced programmed cell death due to MK 886: cells ("soil") "trump" agent ("seed").

Micromolar concentrations of the five-lipoxygenase inhibitor, MK 886 induce a "type 1" (apoptotic, extrinsic, death domain, receptor-dependent, caspase-positive) form of programmed cell death in Bcl-2-positive U937 human monoblastoid and HL-60 myeloid leukemia cells. A "type 2" (intrinsic, mitochondria-dependent, autophagic, in some examples caspase-negative (Panc-1)) form is induced in Panc-1 pancreatic and PC3 prostate cell lines. The latter two lines from epithelial-derived solid human cancers are Bcl-2-negative. Micromolar MK 886 induces an acute rise in Ca2+ in washed, Ca2+-poor U937 and HL60 cells in Ca2+ and Mg2+-free Hank's buffer. In U937 cells, much of the increase, or more properly redistribution, is nuclear in location (HL-60 not tested). No MK-886-induced acute Ca2+ increase developed in Panc-1 or PC3 cells. Bcl-2-positive HeLa cervical cancer cells exhibited an acute MK 886-induced increase in Ca2+. In the U937, PC3 and Panc-1 cells examined, MK-886 rapidly increased oxidative stress and decreased mitochondrial membrane potential, indicating that neither event is directly determinative for the altered distribution of Ca2+ or the form of PCD observed. Inhibition of increased U937 Ca2+ by the anti-oxidant, N-acetyl-L-cysteine, the effects of inhibitors of mitochondrial function including antimycin A, atractyloside, cyclosporin A, the L/N channel blocker loperamide, the intracellular chelator BAPTA and 2 agents, HA-14 and 3-methyl-antimycin A3 that impair Bcl-2 function further define these events. These differences in the Ca2+ response and possibly also the form of PCD that results may depend upon the presence of Bcl-2 or a related protein participating in a juxta-nuclear / nuclear Ca2+ ion channel. The role of mitochondria, the mechanism by which increased oxidative stress initiates the rapid release of Ca2+ from intracellular, possibly juxta-nuclear / nuclear sites or its redistribution to U937 Ca2+ nuclei, and whether this "signal" or possibly even ROS themselves mandate the type of PCD observed, presumably by differential modulation of transcription, remain to be determined. Lastly, these results demonstrate that, as might be expected, "soil" (cell type) trumps "seed" (inciting agent)".

Acute changes in U937 nuclear Ca2+ preceding type 1 "apoptotic" programmed cell death due to MK 886.

BACKGROUND: MK 886, a 5-lipoxygenase inhibitor, induces a type 1 "apoptotic" form of programmed cell death in Bcl-2-positive U937 monoblastoid cells. In Ca2+-depleted, nonpermeabilized U937 cells studied with MK 886 in a Ca2+-free medium, an acute increase in Ca2+ occured within 10 to 20 seconds, detected with fura-2 measured with a spectrofluorimeter. METHODS AND RESULTS: The increased fluorescence was nuclear in location, as judged by confocal microscopy. The antioxidant, N-acetyl-L-cysteine, three agents that inhibit mitochondrialfunction at identified sites, antimycin A, atractyloside and cyclosporin A, the L/N-channel inhibitor, loperamide and BAPTA, an intracellular Ca+ chelator preloaded into cells each reduced the extent or prevented the acute MK 886-induced rise in Ca2+, as determined by radiometric detection. Rhodamine-2, a more selective mitochondrial Ca2+ probe, provided no evidence for nuclear Ca2+ originating from that extra-nuclear site or from the endoplasmic reticulum. With 2', 7'-dichloro-dihydrofluorescein-labelled cells to detect reactive oxygen species, MK 886 increased the initial fluorescent signal from a number of intracellular, largely extra-nuclear sites, including mitochondria. Two chemicals that inhibit the function of Bcl-2, HA14-1 and 2-methyl-antimycin A3, reduced the Ca2+ response to MK 886, if pre-incubated with the Bcl-2-positive U937 cells at 37 degrees C for several hours. MK 886 was previously shown to induce reactive oxygen species and a fall in mitochondrial membrane potential in both Bcl-2-positive U937 and in Bcl-2-negative PC-3 prostate and panc-1 pancreatic cancer cells. The latter solid tumor cells undergo an atypical "type 2" PCD without an acute rise in nuclear Ca2+. CONCLUSION: These results are consistent with an MK 886-induced increase of reactive oxygen species from intra-cellular sites including mitochondria which release Ca2+ located primarily at or near nuclei. These events may involve Bcl-2 participating in some form of Ca2+ channel and nuclear Ca2+ binding proteins undergoing conformational changes due to reactive oxygen species. Reasons for the different PCD responses in Bcl-2 positive lympho-hematopoietic compared to Bcl-2-negative solid cancer cell lines, respectively with and without the induced nuclear Ca2+ signal, remain to be defined.

Regulation of butyrate uptake in Caco-2 cells by phorbol 12-myristate 13-acetate.

Butyrate and the other short-chain fatty acids (SCFAs) are the most abundant anions in the colonic lumen. Also, butyrate is the preferred energy source for colonocytes and has been shown to regulate colonic electrolyte and fluid absorption. Previous studies from our group have demonstrated that the HCO(3)(-)/SCFA(-) anion exchange process is one of the major mechanisms of butyrate transport across the purified human colonic apical membrane vesicles and the apical membrane of human colonic adenocarcinoma cell line Caco-2 and have suggested that it is mainly mediated via monocarboxylate transporter-1 (MCT-1) isoform. However, little is known regarding the regulation of SCFA transport by various hormones and signal transduction pathways. Therefore, the present studies were undertaken to examine whether hydrocortisone and phorbol 12-myristate 13-acetate (PMA) are involved in a possible regulation of the butyrate/anion exchange process in Caco-2 cells. The butyrate/anion exchange process was assessed by measuring a pH-driven [(14)C]butyrate uptake in Caco-2 cells. Our results demonstrated that 24-h incubation with PMA (1 microM) significantly increased [(14)C]butyrate uptake compared with incubation with 4alphaPMA (inactive form). In contrast, incubation with hydrocortisone had no significant effect on butyrate uptake in Caco-2 cells compared with vehicle (ethanol) alone. Induction of butyrate uptake by PMA appeared to be via an increase in the maximum velocity (V(max)) of the transport process with no significant changes in the K(m) of the transporter for butyrate. Parallel to the increase in the V(max) of [(14)C]butyrate uptake, the MCT-1 protein level was also increased in response to PMA incubation. Our studies demonstrated that the butyrate/anion exchange was increased in response to PMA treatment along with the induction in the level of MCT-1 expression in Caco-2 cells.

Increased cytosol Ca(2+) and type 1 programmed cell death in Bcl-2-positive U937 but not in Bcl-2-negative PC-3 and Panc-1 cells induced by the 5-lipoxygenase inhibitor MK 886.

MK 886, an arachidonic acid-related analog which inhibits the enzyme, 5-lipoxygenase by an indirect mechanism involving the 5-lipoxygenase activating protein, rapidly increased U937 cytosol Ca(2+), much of which localized around the cell nuclei. Five-lipoxygenase activity was not directly involved since the direct redox-dependent 5-LPOx inhibitor, SC-41661A did not increase Ca(2+). U937 cells subsequently undergo classic type 1 programmed cell death. At least initially the ionized calcium originates from internal stores. Coincident with the rise in U937 ionized calcium, MK 886 rapidly increased reactive oxygen species and reduced mitochondrial membrane potential, as judged by several fluorescent probes. The Ca(2+) response of myeloid leukemia-derived HL-60 cells to MK 886 was similar and both cell lines express Bcl-2 protein. Bcl-2-negative Panc-1 and PC-3 cells did not respond to MK 886 with a Ca(2+) signal but did develop oxidative stress and a decline in mitochondrial membrane potential; these events are thought to contribute to the inhibition of cell proliferation and induction of a type 2 PCD. In addition to its marked inhibition of Bcl-2 mRNA synthesis, an interesting hypothesis is that MK 886, serving as a low molecular weight ligand, either by direct or indirect inhibition of U937 Bcl-2 protein function, possibly related to an ion channel activity, alters the distribution of intracellular, possibly nuclear Ca(2+), thereby promoting the development of type 1 programmed cell death.

A response of Panc-1 cells to cis-platinum, assessed with a cDNA array.

BACKGROUND: The problem posed by the lack of response of cells in most solid cancers to current chemotherapy generally remains intractable. MATERIALS AND METHODS: The use of cDNA arrays represents one global approach to identifying reasons for this failure. A messenger RNA response of pancreatic cancer (Panc-1) cells after culture for 24 hours with 12 microM cis-platinum was analyzed with a commercial cDNA array. RESULTS: Major drug-induced events included inhibition of messenger RNAs associated with cell proliferation and up-regulation of generally countervailing DNA repair, cellular stress, heat shock protein, glutathione stress-related and multiple drug resistance enzyme messenger RNAs, accompanied by a limited programmed cell death response. CONCLUSION: Induction of widespread normal stress-induced countervailing mRNAs by comparatively non-selective agents such as cis-platinum strongly biases against a successful therapeutic outcome. This paradoxical result of a therapeutic intent provides a further compelling argument for the use of specifically-targeted therapy such as growth factor receptor, tyrosine kinase and other discretely focused agents, probably employed in combinations based on expression of their targets in an individual patient's cancer, as identified by cDNA or proteonomic arrays.

Differential regulation of the expression of Na(+)/H(+) exchanger isoform NHE3 by PKC-alpha in Caco-2 cells.

Na(+)/H(+) exchange (NHE) activity has been shown to be regulated by various external signals and protein kinases in many tissues and cell types. A family of six NHE isoforms has been identified. Three isoforms, NHE1, NHE2, and NHE3, have been shown to be expressed in the human intestine. The present studies were designed to study regulation of these human NHE isoforms by the alpha-isoform of protein kinase C (PKC) in the Caco-2 cell line. The mRNA levels of the NHE isoforms in Caco-2 cells were initially measured by a semiquantitative RT-PCR technique in response to PKC downregulation by long-term exposure to 1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 24 h. PKC downregulation resulted in an approximately 60% increase in the mRNA level for NHE3, but not for NHE1 or NHE2. Utilizing dichlorobenzimidazole riboside, an agent to block the synthesis of new mRNA, we demonstrated that the increase in the NHE3 mRNA in response to downregulation of PKC was predominantly due to an increase in the rate of transcription, rather than a decrease in the NHE3 mRNA stability. Consistent with the mRNA results, our data showed that amiloride-sensitive (22)Na(+) uptake was increased after incubation of Caco-2 cells with 1 microM TPA for 24 h. To elucidate the role of PKC-alpha, an isoform downregulated by TPA, the relative abundance of NHE isoform mRNA levels and the apical NHE activity were assessed in Caco-2 cells over- and underexpressing PKC-alpha. Our results demonstrated that NHE3, but not NHE1 or NHE2, mRNA was downregulated by PKC-alpha and that apical NHE activity was higher in cells underexpressing PKC-alpha and lower in cells overexpressing PKC-alpha than in control cells. In conclusion, these data demonstrate a differential regulation of NHE3, but not NHE2 or NHE1, expression by PKC in Caco-2 cells, and this regulation appears to be predominantly due to PKC-alpha.

Mechanism(s) of chloride transport in human distal colonic apical membrane vesicles.

Previous studies from our laboratory have demonstrated the presence of an electroneutral Cl-/HCO3- exchange process across the human proximal colonic apical membrane vesicles (AMV). However, very little is known about the mechanism(s) of chloride transport in the apical membrane of the human distal colon. Utilizing AMV purified from organ donor distal colonic mucosa and a rapid Millipore filtration technique, the mechanisms of 36Cl- uptake into these vesicles were examined. Outwardly directed OH and HCO3 gradients markedly increased the uptake of 36Cl- into these vesicles, demonstrating a transient accumulation over the equilibrium uptake. Voltage clamping in the presence of K+/valinomycin reduced the OH and HCO3- gradient-stimulated 36Cl- uptake into these vesicles by approximately 30% indicating that the conductive Cl- uptake pathway was present in these vesicles along with the electroneutral exchange process. Under voltage-clamped conditions, the inhibitors the bicarbonate transporters, DIDS and SITS (1 mM), inhibited OH and HCO3- gradient-stimulated 36Cl- uptake by approximately 50%. Acetazolamide showed small but significant inhibition of chloride uptake. Amiloride, bumetanide, and furosemide failed to inhibit 36Cl- uptake. Chloride uptake into these vesicles exhibited saturation kinetics with an apparent Km for chloride of 16.7 mM and a Vmax of 5.9 nmol/mg/15 sec. Chloride, acetate, nitrate, but not sulfate (50 mM each), inhibited 5 mM 36Cl- uptake. Inwardly directed gradients of Na+, K+ or both together did not stimulate chloride uptake into these vesicles indicating that the uptake of Cl- and Na+ in human distal colonic AMV does not involve Na-Cl or Na-K-2Cl cotransport. In conclusion, these studies demonstrate that Cl- transport across the apical membranes of human distal colon involves both conductive pathway and electroneutral Cl-/HCO3- (OH-) exchange processes. In view of our previous demonstration of a Na+/H+ exchange process in these AMV, we propose that the operation of dual ion exchange mechanisms of Na+/H+ and Cl-/HCO3- is the primary mode of electroneutral NaCl absorption across the apical membranes of the enterocytes of the human distal colon.

Evidence for the existence of a distinct SO(4)(--)-OH(-) exchange mechanism in the human proximal colonic apical membrane vesicles and its possible role in chloride transport.

Recent studies have demonstrated that mutations in human downregulated in adenoma gene (DRA) result in congenital chloride diarrhea (CLD), and that DRA may be involved in chloride transport across the intestinal apical domains. DRA is highly homologous to sulfate transporters, but not to any member of the anion exchanger gene family (AEs). Our previous studies have characterized the existence of a distinct Cl(-)-OH(-) (HCO(3)(-)) exchanger, with minimal affinity for sulfate in the human colonic apical membrane vesicles (AMV). However, the mechanism(s) of sulfate movement across the colonocyte plasma membranes in the human colon is not well understood. Current studies were undertaken to elucidate sulfate transport pathways in AMVs of human proximal colon. Purified AMV and rapid filtration (35)SO(4)(--) uptake techniques were used. Our results demonstrate the presence of a pH gradient-driven carrier-mediated SO(4)(--)-OH(-) exchange process in the human proximal colonic luminal membranes based on the following: a marked increase in the SO(4)(--) uptake in the presence of an outwardly directed OH(-) gradient; a significant inhibition of SO(4)(--) uptake by the membrane anion transport inhibitor, DIDS; demonstration of saturation kinetics (K(m) for SO(4)(--): 0.80 +/- 0.17 mM and Vmax 649 +/- 74 pmol/mg protein/10 sec); competitive inhibition of SO(4)(--)-OH(-) exchange by oxalate; SO(4)(--) uptake was insensitive to alterations in the membrane potential; and inwardly directed Na(+) gradient under non-pH gradient conditions did not stimulate SO(4)(--) uptake. SO(4)(--) uptake was significantly inhibited by increasing concentrations of chloride (1-10 mM) in the incubation media with a K(i) for Cl(-) of 9.3 +/- 1.4 mM. In contrast, OH(-)/HCO(3)(-) gradient-driven (36)Cl(-) uptake into these vesicles was unaffected by increasing concentrations of sulfate (10-50 mM). The above data indicate that two distinct transporters may be involved in SO(4)(--) and Cl(-) transport in the human intestinal apical membranes: an anion exchanger with high affinity for SO(4)(--) and oxalate but low affinity for Cl(-), and a distinct Cl(-)-OH(-) (HCO(3)(-)) exchanger with low affinity for SO(4)(--).

Sulfate and chloride transport in Caco-2 cells: differential regulation by thyroxine and the possible role of DRA gene.

The current studies were undertaken to establish an in vitro cellular model to study the transport of SO and Cl(-) and hormonal regulation and to define the possible function of the downregulated in adenoma (DRA) gene. Utilizing a postconfluent Caco-2 cell line, we studied the OH(-) gradient-driven (35)SO and (36)Cl(-) uptake. Our findings consistent with the presence of an apical carrier-mediated (35)SO/OH(-) exchange process in Caco-2 cells include: 1) demonstration of saturation kinetics [Michaelis-Menten constant (K(m)) of 0.2 +/- 0.08 mM for SO and maximum velocity of 1.1 +/- 0.2 pmol x mg protein(-1) x 2 min(-1)]; 2) sensitivity to inhibition by DIDS (K(i) = 0.9 +/- 0.3 microM); and 3) competitive inhibition by oxalate and Cl(-) but not by nitrate and short chain fatty acids, with a higher K(i) (5.95 +/- 1 mM) for Cl(-) compared with oxalate (K(i) = 0.2 +/- 0.03 mM). Our results also suggested that the SO/OH(-) and Cl(-)/OH(-) exchange processes in Caco-2 cells are distinct based on the following: 1) the SO/OH(-) exchange was highly sensitive to inhibition by DIDS compared with Cl(-)/OH(-) exchange activity (K(i) for DIDS of 0.3 +/- 0.1 mM); 2) Cl(-) competitively inhibited the SO/OH(-) exchange activity with a high K(i) compared with the K(m) for SO, indicating a lower affinity for Cl(-); 3) DIDS competitively inhibited the Cl(-)/OH(-) exchange process, whereas it inhibited the SO/OH(-) exchange activity in a mixed-type manner; and 4) utilizing the RNase protection assay, our results showed that 24-h incubation with 100 nM of thyroxine significantly decreased the relative abundance of DRA mRNA along with the SO/OH(-) exchange activity but without any change in Cl(-)/OH(-) exchange process. In summary, these studies demonstrated the feasibility of utilizing Caco-2 cell line as a model to study the apical SO/OH(-) and Cl(-)/OH(-) exchange processes in the human intestine and indicated that the two transporters are distinct and that DRA may be predominantly a SO transporter with a capacity to transport Cl(-) as well.

Human intestinal anion exchanger isoforms: expression, distribution, and membrane localization.

A family of anion exchangers (AEs) including AE1, AE2 and AE3 has been described. AE3 gene has been shown to encode two alternatively spliced isoforms termed as bAE3 (brain subtype) and cAE3 (cardiac subtype). The identity of the AE(s) involved in the human intestinal NaCl absorption is not fully understood. Current studies were undertaken to identify the AE isoforms expressed in the human intestine, to define their regional and vertical axis (crypt vs. surface cells) distribution, and to elucidate their membrane localization in the epithelial cells along the entire length of the human intestine. Our studies utilizing reverse transcription (RT)-PCR with total RNA extracted from pinch biopsies from various regions of the human intestine demonstrate that AE2 and bAE3 but not AE1 or cAE3 were expressed in all the regions of the human intestine. Utilizing in situ RT-PCR, we demonstrated that the message of AE2 was expressed throughout the vertical surface--crypt axis of the colon. Our Western blotting studies demonstrated that AE2 and bAE3 are localized to the basolateral but not the apical membranes of the intestinal epithelial cells from the human ileum and colon. In conclusion, our results demonstrated that in the human intestine, AE2 and bAE3, but not AE1 or cAE3, are expressed throughout the tract with the highest expression in the colon compared to the ileum and jejunum. Both the isoforms were found to be localized to the basolateral but not the apical membranes of the epithelial cells. We speculate that, in the human intestine, AE2 and bAE3 may be the 'housekeeping' isoforms, and the apical AE, the potential candidate for chloride absorption, remains to be identified.

Detection of Cl--HCO3- and Na+-H+ exchangers in human airways epithelium.

Molecular species of the Na(+)-H(+) exchanger (NHE) and anion exchanger (AE) gene families and their relative abundance in the human airway regions were assessed utilizing RT-PCR and the RNase protection assay, respectively. Organ donor lung epithelia from various bronchial regions (small, medium, and large bronchi and trachea) were harvested for RNA extraction. Gene-specific primers for the human NHE and AE isoforms were utilized for RT-PCR. Our results demonstrated that NHE1, AE2, and brain AE3 isoforms were expressed in all regions of the human airway, whereas NHE2, NHE3, AE1, and cardiac AE3 were not detected. RNase protection studies for NHE1 and AE2, utilizing glyceraldehyde-3-phosphate dehydrogenase as an internal standard, demonstrated that there were regional differences in the NHE1 mRNA levels in human airways. In contrast, the levels of AE2 mRNA remained unchanged. Differential regional expression of NHE1 isoform may be related to a higher acid load in the tracheal epithelial cells than in epithelia of distal airways. Fluctuations in PCO(2) during inspiration and expiration are probably larger in the tracheal lumen than in the lumen of distal airways with associated larger swings in intracellular pH with each respiratory cycle. Immunohistochemical staining for AE2 protein demonstrated localization to the epithelial cells of human bronchial mucosa.

A genomic response of H-358 bronchiolar carcinoma cells to MK 886, an inhibitor of 5-lipoxygenase, assessed with a cDNA array.

Incomplete programmed cell death is one explanation for the escape of cancer cells from therapy. Inhibitors of the enzyme 5-lipoxygenase reduce proliferation and initiate programmed cell death in many different types of malignantly transformed cells. The 5-lipoxygenase inhibitor, MK 886. induces an atypical form of programmed cell death in H-358 bronchiolar lung cancer cells. A genomic response of H-358 cells after 24 hr of culture at a 40 uM concentration that inhibited proliferation was analyzed with a Clontech human cDNA array containing 588 cDNAs corresponding to identified genes. The data grouped into 3 major categories and initial conclusions regarding countervailing, cellular stress, programmed cell death, DNA damage and repair mRNA-responses as possible reasons for escape from the antiproliferative response are discussed. The use of cDNA arrays to estimate the extent to which malignantly transformed cells respond to therapy or why they do not and so infer prognosis and identify possible therapeutic modifications is indicated.

Widespread countervailing genomic responses induced by chemotherapy or radiation as a cause of therapeutic failure.

If chemotherapy or ionizing radiation induce widespread genomic responses tending to circumvent or antagonize their ability to kill malignant cells, an additional cause for therapeutic failure would be suggested. There is evidence that some agents evoke extensive countervailing genomic activity, the nature and extent of which can be assessed with the use of 'gene chips'.

Evidence for a Na+-H+ exchange across human colonic basolateral plasma membranes purified from organ donor colons.

The mechanism(s) of electrolyte transport across the human colonic contraluminal domain is not well understood. Current studies were undertaken to develop a technique for the isolation and purification of the human colonic basolateral membrane vesicles (BLMV) and to examine the presence of a Na+-H+ exchange process in these membranes. BLMV were purified from mucosal scrapings of organ donor proximal colons utilizing a Percoll density gradient centrifugation technique, and Na+ transport was examined utilizing a rapid filtration, technique. Our data demonstrate that purified basolateral membranes were enriched 10- to 11-fold in Na+, K+-ATPase activity compared to crude homogenate. Results consistent with the Na+-H+ exchange in BLMV are as follows: (1) an outwardly directed H+ gradient stimulated 22Na uptake; (2) 22Na uptake was markedly inhibited by EIPA and amiloride; (3) H+-gradient-stimulated 22Na uptake was not inhibited by bumetanide, SITS, DIDS, acetazolamide, phenamil and benzamil; (4) 22Na uptake was voltage insensitive; (5) 22Na uptake demonstrated saturation kinetics; (6) 22 Na uptake was markedly inhibited by Na+ and Li+ but was unaffected by N-methyl glucamine+, choline+, and NH4+. Immunoblotting studies demonstrated this Na+-H+ exchanger isoform to be represented by NHE1. In conclusion, a technique has been established for the purification of functional human proximal colonic BLMV, and an electroneutral Na+-H+ exchange process has been demonstrated in these membranes.

Expression of the Na+/H+ and Cl-/HCO-3 exchanger isoforms in proximal and distal human airways.

Recent studies have indicated the presence of Na+/H+ and Cl-/HCO-3 exchange activities in lung alveolar and tracheal tissues of various species. To date, the identity of the Na+/H+ (NHE) and Cl-/HCO-3 (AE) exchanger isoforms and their regional distribution in human airways are not known. Molecular species of the NHE and AE gene families and their relative abundance in the human airway regions were assessed utilizing RT-PCR and the RNase protection assay, respectively. Organ donor lung epithelia from various bronchial regions (small, medium, and large bronchi and trachea) were harvested for RNA extraction. Gene-specific primers for the human NHE and AE isoforms were utilized for RT-PCR. Our results demonstrated that NHE1, AE2, and brain AE3 isoforms were expressed in all regions of the human airways, whereas NHE2, NHE3, AE1, and cardiac AE3 were not detected. RNase protection studies for NHE1 and AE2, utilizing glyceraldehyde-3-phosphate dehydrogenase as an internal standard, demonstrated that there were regional differences in the NHE1 mRNA levels in human airways. In contrast, the levels of AE2 mRNA remained unchanged. Differential expression of these isoforms in the human airways may have functional significance related to the airway absorption and secretion of electrolytes.

Altered oncogene, tumor suppressor and cell-cycle gene expression in PANC-1 cells cultured with the pleiotrophic 5-lipoxygenase inhibitor, MK886, assessed with a gene chip.

We describe a genomic response of mRNAs associated with a subset of oncogenes, tumor suppressor and cell cycle-related genes in proliferating human Panc-1 pancreatic cancer cells after 24 hours of culture with MK886, a pleotrophic 5-lipoxygenase inhibitor. Ninety-eight of these cDNAs are represented in one of the sub-arrays included in the Clontech Human cDNA Expression Array. In this initial analysis, control cells exhibited apparent widespread low levels of disparate mRNA synthesis. In cells cultured with 40 microM MK886 for 24 hr, while most expressed genes, including a number of specific proliferation-enhancing genes such as c-myc were inhibited, 19 other ones including some countervailing genes including tyrosine SRC protein kinase, cyclins B1 and D1, CDC25B phosphatase and 40s ribosomal S19, amounting to 19 percent of the cDNAs resident on the chip were up-regulated at > 1.10 experimental/control values. Therapy-induced activation of compensatory proliferative genomic responses provides an additional explanation why malignant cells can fail therapy. Among their many future uses, gene chips clearly will be an extremely powerful tool for identifying relationships between the hierarchical linear and non-linear control and implementation-related cellular events and for identifying potential molecular targets tor cancer therapy.

Five-lipoxygenase inhibitors reduce Panc-1 survival: the mode of cell death and synergism of MK886 with gamma linolenic acid.

The 5-lipoxygenase inhibitors ETYA, SC41661A and MK886 reduced the proliferation and viability of Panc-1 human pancreatic cancer cells. The extent of inhibition depended upon drug concentration, and with continued culture, cells detached and stained with trypan blue. Although results from flow cytometry were those associated with programmed cell death, despite repeated attempts, no DNA laddering consistent with its later stages was detected, and studies with the TUNEL assay were negative. Light and electron microscopy of cells cultured with SC41661A provided morphologic evidence of a population of "dark" cells and of an incompletely expressed type 1 programmed cell death including margination of chromatin at the nuclear membrane and by consolidation and degeneration of cytoplasmic organelles, along with extensive vacuolization. Cells cultured with MK886 exhibited compact "dark" cells and an unusual cytoplasmic mode of cell death characterized by vacuolization and widely separated smooth internal membranes without diagnostic nuclear changes. This is in marked contrast to the extensive type 1 PCD induced by 5-lipoxygenase inhibitors cultured with human U937 monoblastoid cells. On balance, the response of Panc-1 cells to MK886 suggests expression of a variant type 2 (autophagic) cellular suicide, although some contribution from components of a "cytoplasmic" (type 3?) form of non-necrotic cell death may also be considered. In a European clinical trial, gamma linolenic acid, a polyunsaturated fatty acid that generates free radicals has been combined with 5-fluorouracil as chemotherapy for pancreatic cancer. Panc-1 cell proliferation was insensitive to inhibition by several chemotherapeutic agents employed clinically, including 5-fluorouracil, cisplatin or gemcitabine and only somewhat sensitive to GLA. When gamma linolenic acid was combined with MK886, the more effective of the two 5-lipoxygenase inhibitors, a synergistic reduction in Panc-1 cell number and viability occurred.