Sally L. D. MacKay

Transcribed heteroplasmic repeated sequences in the porcine mitochondrial DNA D-loop region

The mitochondrial D-loop region of the pig, Sus scrofa, was found to be several hundred base pairs larger than the corresponding region in cow, a related artiodactyl species, primarily because of an insertion containing the tandemly repeated sequence CGTGCGTACA. Porcine mitochondrial DNA from the tissue of a single animal exhibits a large population of length polymorphs, each member of which may have as few as 14 or as many as 29 of these repeat units. This intracellular variability may be due to the repeated and self-complementary properties of this sequence, which would favor mispairing and lead to replication slippage. The repeat domain is unusual in that symmetry properties suggest it may assume alternative conformations including cruciforms and left-handed (Z) DNA. It also appears to be the longest known, naturally occurring, alternating purine-pyrimidine sequence. In order to understand the functional significance of this heteroplasmic domain that potentially disrupts a key regulatory region in the mitochondrial genome, RNA and DNA mapping studies were conducted which located this region between the H-strand replication origin and the putative L-strand transcriptional start site. H-strand RNA analysis demonstrated that this heteroplasmic region is transcribed and, therefore, that priming for H-strand DNA replication in mitochondria is independent of the primer RNA length or secondary structure.

A matrix metalloproteinase inhibitor prevents processing of tumor necrosis factor α (TNFα) and abrogates endotoxin-induced lethality

Excessive tumor necrosis factor α (TNFα) production in response to Gram-negative bacteremia or endotoxemia can often lead to hypotension, shock, and increased mortality. Current approaches used to block the deleterious effects of exaggerated TNFα production rely on monoclonal antibodies or immunoadhesins that bind TNFα and thus prevent the interaction with its cellular receptors. This report examines whether a previously described inhibitor of matrix metalloproteinases, GM-6001, can inhibit TNFα processing and release and attenuate endotoxin-induced mortality. In human peripheral blood mononuclear cells stimulated in vitro with 1 μg/mL endotoxin, GM-6001 at concentrations <5 μg/mL blocked release of TNFα, but did not affect the release of either IL-1β or IL-6. GM-6001 also inhibited the release of soluble TNF receptor (p75) from peripheral blood mononuclear cells stimulated with endotoxin and/or TNFα. To confirm the role of secreted TNFα in endotoxic shock-induced mortality, C57BL/6 mice were challenged with either endotoxin alone (500 μg/mouse) or endotoxin (100 ng/mouse) plus D-galactosamine (8 mg/mouse). GM-6001 pretreatment (100 mg/kg) significantly attenuated the 90-minute plasma TNFα response in both models and improved survival in mice treated with low-dose endotoxin plus D-galactosamine. However, plasma IL-1β and IL-6 concentrations at 90 min after endotoxin treatment were unaffected by GM-6001 following lethal endotoxin challenge, confirming the in vivo specificity of this matrix metalloproteinase inhibitor for TNFα processing. These findings demonstrate that a novel inhibitor of matrix metalloproteinases can prevent the release of TNFα both in vitro and in vivo, and can abrogate the harmful sequelae of endotoxemic shock.

Caspase-3–Dependent Organ Apoptosis Early After Burn Injury

OBJECTIVE To examine the role played by endotoxin, tumor necrosis factor-alpha (TNF-alpha), and caspase-3 in the increased apoptosis seen in solid organs in the early period after a burn injury. SUMMARY BACKGROUND DATA Burn injury is often associated with immune suppression. Bacterial translocation and systemic endotoxemia have been reported after a burn injury, and caspase-3 activation due to TNF-alpha and Fas ligand (FasL) are presumed to initiate apoptosis. We hypothesized that endotoxin-induced TNF-alpha expression and caspase-3 activation could be the stimulus for the apoptosis after burn injury. METHODS A 20% full-thickness scald burn was used in C57BL/6 mice. Three hours after burn injury, tissue samples were obtained from the thymus, lung, liver, and spleen. Lipopolysaccharide-nonresponsive (C3H/HeJ) and TNFalpha null B6x129tnf-/- mice were also used. To detect apoptosis, hematoxylin and eosin stain, in situ TUNEL, DNA extraction, and gel electrophoresis were all performed. Caspase-3 activity and TNF-alpha and FasL mRNA were also measured. RESULTS Increased apoptosis and caspase-3 activity were observed in the thymus and spleen 3 hours after burn injury but were not seen in liver or lung. In the thymus and spleen, increased expression of FasL mRNA was also observed, whereas increased TNF-alpha mRNA was not. Increased apoptosis in thymus and spleen were also observed in C3H/HeJ and B6x129tnf-/- mice after burn injury. An inhibitor of the caspase-3 (Z-VAD-fmk) reduced apoptosis in both thymus and spleen. CONCLUSIONS In the early period after a burn injury, increased apoptosis is observed primarily in the lymphoid organs and is independent of endotoxin or TNF-alpha. The increased caspase-3 activity in thymus and spleen contributes to apoptosis in these organs.

Colon Cancer Cells That Are Not Growth Inhibited by TGF-β Lack Functional Type I and Type II TGF-β Receptors

OBJECTIVE The authors determined the molecular mechanisms for the failure of transforming growth factor-beta (TGF-beta) to inhibit the growth of SW1116 and SW48 colon cancer cell lines. BACKGROUND Transforming growth factor-beta is a bifunctional regulator of cell growth that typically stimulates proliferation of mesenchymal cells, but inhibits proliferation of normal epithelial cells. In the colon, TGF-beta appears to arrest proliferation of enterocytes as they leave the intestinal crypt and move to the villus tip. Transforming growth factor-beta actions are mediated by binding to heteromeric complexes of type I and type II TGF-beta receptors. Loss of TGF-beta responsiveness may contribute to uncontrolled cell growth and cancer. METHODS The effects of TGF-beta 1 on DNA synthesis were measured by incorporation of tritiated thymidine into DNA of cultures of moderately differentiated adenocarcinoma (SW48) and poorly differentiated adenocarcinoma (SW1116) colon cell lines and a mink lung epithelial cell line (CCL-64). The effects of TGF-beta on the expression of c-myc, TGF-alpha, and TGF-beta in SW48 cells, SW1116 cells, and CCL-64 cells (c-myc only) were measured by Northern blot analysis. Expression of TGF-beta receptors in the cell lines was measured using competitive binding assays, receptor affinity labelling techniques, and reverse transcriptase-polymerase chain reaction. RESULTS Incubation with TGF-beta 1 (50 ng/mL) did not decrease serum-stimulated uptake of [3H]-thymidine into actively growing cultures of SW48 or SW1116 cells, but suppressed DNA synthesis of actively growing CCL-64 cells by 90%. Similarly, incubation with TGF-beta 1 (12 ng/mL) for 4 hours did not substantially alter the mRNA levels of c-myc, TGF-alpha, and TGF-beta 1 in either colon tumor cell line, although levels of c-myc mRNA in CCL-64 cells were reduced by TGF-beta 1 treatment. Competitive displacement of [125I]-TGF-beta 1 binding detected high levels (16,500 TGF-beta receptors per cell) of specific, high-affinity (200 pmol/L half-displacement) TGF-beta receptors on CCL-64 cells. In marked contrast, very low levels of TGF-beta 1 binding to SW1116 cells (250 receptors per cell) and SW48 cells (260 receptors per cell) were detected. Autoradiograms of CCL-64 cells affinity labelled with [125I]TGF-beta 1 revealed the presence of type I, type II, and type III TGF-beta receptors. No TGF-beta receptors were identified on SW1116 cells, and only very low levels of the nonsignaling type III TGF-beta receptors were detected on SW48 cells. Reverse transcriptase-polymerase chain reaction amplification detected mRNAs for type I, type II, and type III TGF-beta receptors in CCL-64 cells. SW48 cells, and SW1116 cells. CONCLUSIONS These results suggest that the lack of growth inhibition by TGF-beta in SW48 and SW1116 colon cancer cells may be caused by a lack of expression of functional TGF-beta receptors.

Discordant tumor necrosis factor-α superfamily gene expression in bacterial peritonitis and endotoxemic shock

BACKGROUND Tumor necrosis factor-alpha (TNF-alpha) is a member of a large family of predominantly homotrimeric type II membrane-associated proteins with both proinflammatory and apoptosis-inducing properties. Although TNF-alpha expression has been studied extensively, little is known about the expression of other members of the TNF-alpha superfamily during acute inflammatory processes. METHODS TNF-alpha, Fas ligand (FasL), and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) messenger RNA (mRNA) expression were examined in liver, lung, spleen, and kidney after either a cecal ligation and puncture or endotoxemic shock with use of semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS Cecal ligation and puncture increased TNF-alpha mRNA in lung and liver (both P < .05) within 3 hours, which was paralleled by increased FasL mRNA. In the spleen TNF-alpha and FasL mRNA significantly declined (both P < .05). In contrast to TNF-alpha and FasL, TRAIL mRNA levels were unchanged in all organs except lung, where it was reduced at 24 hours (P < .05). Endotoxemic shock also increased lung TNF-alpha and FasL mRNA levels (both P < .05). CONCLUSIONS In acute inflammatory processes TNF-alpha and FasL mRNA increase concordantly in several solid organs. In contrast, TRAIL mRNA levels do not consistently change during these acute inflammatory processes, suggesting that its expression is under independent and discordant regulatory control.

Transient human gene therapy: a novel cytokine regulatory strategy for experimental pancreatitis.

OBJECTIVE The purpose of this study was to evaluate the ability to transfect a murine pancreas with a human cytokine regulatory gene (interleukin-10 [IL-10]) and examine the duration of transgene expression, its effect on the normal pancreas, and its antiinflammatory effect during acute pancreatitis. SUMMARY BACKGROUND DATA Interleukin-1beta and tumor necrosis factor-alpha are known detrimental mediators during the progression of acute pancreatitis, and blockade of either cytokine results in decreased severity of pancreatitis and improved survival. Although gene therapy has been proposed as a method to deliver protein-based therapy during a number of conditions, no means of effectively transfecting the pancreas without inducing injury has been developed. METHODS A plasmid-human IL-10 construct (pMP6-hIL-10) complexed with cationic liposomes was administered by single intraperitoneal injection to healthy mice. Effective transfection (reverse transcriptase-polymerase chain reaction for hIL-10 mRNA), transfected cell type (in situ polymerase chain reaction for hIL-10 DNA), and the effect on the normal pancreas were determined. Additional animals were transfected to determine the effects of this regulatory gene on the severity of pancreatitis. RESULTS Nearly 80% of all pancreatic cells expressed human DNA that was subsequently transcribed into mRNA through day 14. The transfection event had no effect on amylase, lipase, or pancreatic histologic appearance. Successful transfection could attenuate subsequently induced pancreatitis (all parameters p < 0.05). CONCLUSIONS Transient transfection of a human IL-10 gene can be accomplished into all cell types of murine pancreata using a plasmid/ liposome vector. The DNA is effectively transcribed into intact mRNA and does not cause inflammation or acinar cell damage. Transfer of this cytokine regulatory gene decreases the severity of pancreatitis, demonstrating a benefit of gene therapy during this acute inflammatory process.

Transfection of the Type Ii Tgf-β Receptor Into Colon Cancer Cells Increases Receptor Expression, Inhibits Cell Growth, and Reduces the Malignant Phenotype

OBJECTIVE To determine if transfection of SW48 colon cancer cells with the type II transforming growth factor-beta (TGF-beta) receptor restores growth inhibition and reverses the in vitro and in vivo malignant phenotype. SUMMARY BACKGROUND DATA The authors have previously shown that SW48 colon cancer cells that are replication error positive in both alleles lack functional cell surface TGF-beta type I (RI) and type II (RII) receptors and are insensitive to TGF-beta1-induced growth inhibition. METHODS SW48 cells were stably transfected with the cDNA for the normal type II TGF-beta receptor (RII). Once transfected, the cells were evaluated for in vitro phenotypic changes and in vivo changes in tumor growth. RESULTS Denaturing sequencing gel electrophoresis of the reverse transcriptase-polymerase chain reaction product from SW48 cells revealed that the RII coding sequence contained a single base deletion mutation. When these cells were transfected with normal RII cDNA, Northern and Western blot analyses revealed increased levels of RII mRNA and protein. Affinity labeling techniques revealed that RII-transfected SW48 cells produced functional RI and RII protein. Transfection of SW48 cells also led to changes in cell phenotype, as shown by inhibition of both in vitro growth rate and incorporation of [3H]-thymidine. SW48 cells expressing normal RII also exhibited reduced cloning efficiency in semisolid medium and reduced growth as a xenograft in NOD/LtSz-scid/J mice. CONCLUSIONS The results confirm that RII is a tumor-suppressor protein that is required for TGF-beta-induced growth inhibition in SW48 colon cancer cells.

Application of gene therapy to acute inflammatory diseases.

The application of gene therapy to acute inflammation has not received as much research attention as has the treatment of genetically-based diseases, cancer, and viral infections. However, gene therapy as a drug delivery system offers several theoretical and practical advantages over current protein delivery systems. These include the ability to target therapies to individual tissues or cell types, to locally produce proteins that can act intracellularly or in an autocrine, juxtacrine, or paracrine fashion, and to sustain new protein synthesis for periods up to several weeks after a single administration. Although retrovirus, herpes simplex, and adeno-associated virus have been proposed for gene therapy in cancer and in genetic diseases, nonviral and adenovirus approaches appear most applicable as drug delivery systems due to their rapid onset and short duration of transgene expression. The relative modest transduction efficiencies obtained at present with nonviral approaches, and the inherent inflammatory properties of first-generation adenovirus constructs, however, have limited their usefulness to date. The present review discusses the theoretical and practical benefits of specific gene therapy approaches for the treatment of acute inflammatory diseases, as well as our experiences with liposome:plasmid DNA and adenovirus-based approaches. Although a number of technical and theoretical hurdles remain before it can be evaluated in humans with acute inflammation, gene therapy offers a novel approach for the treatment of acute inflammation, and will likely enter the armamentarium of critical care physicians in the near future.

Cationic liposome-mediated gene transfer during acute pancreatitis: tissue specificity, duration, and effects of acute inflammation☆☆☆

Production of inflammatory cytokines in the pancreas, lung, and liver is believed to play a major role in the development of severe pancreatitis. This tissue-specific production could lend itself to directed anticytokine gene therapy if an appropriate delivery system could be developed. This study was undertaken to examine a novel approach for the delivery of protein-based therapies to the tissues involved during acute pancreatitis. Healthy mice received an intraperitoneal injection of cationic liposomes and a DNA plasmid containing the chloramphenicol acetyltransferase (CAT) reporter gene. Animals were killed at 12 hours and 1, 2, 3, 7, and 14 days with serum, pancreas, lung, and liver harvested. Acute pancreatitis was induced (cerulein, 50 μg/kg/hr intraperitoneally × 4) in additional mice before or after CAT transfection. The presence of pancreatitis was established in all animals by histologic scoring of pancreata and by serum amylase and lipase levels. CAT transfection efficiency was determined by quantitative CAT enzyme activity within tissue homogenates. Animals that received the liposome were successfully transfected with the CAT gene into the pancreas, lungs, and liver. Maximal transfection in each tissue occurred at 12 hours with decreasing CAT activity over the ensuing 14 days. No healthy animals receiving the CAT gene developed elevations in amylase, lipase, or any histologic parameter of pancreatitis. Transfection efficiency in the pancreas was markedly increased by preexisting or delayed induction of pancreatitis, whereas transfection of the lung and liver was increased to a lesser extent. Gene’transfection into the pancreas, liver, and lungs is possible using a cationic liposome delivery system that does not induce pancreatitis or pancreatic inflammation. Pancreatic expression of the gene product is equal to or greater than that of the organs of the reticuloendothelial system and continues at very high efficiency rates during acute pancreafitis.

Inhibition of transforming growth factor alpha stimulation of human squamous cell carcinoma of the head and neck with anti-TGF-α antibodies and tyrphostin

AbstractBackground: Transforming growth factor alpha (TGF-α) and its receptor (EGF-R) may regulate normal and malignant epithelial cell growth by an autocrine mechanism. We investigated the role of TGF-α in regulating head and neck SCC tumor growth. Methods: TGF-α and EGF-R levels were measured in 7 SCC cell lines and 14 SCC biopsies by RIA, Scatchard, and Western analysis. TGF-α autocrine stimulation of DNA synthesis in SCC cell lines was assessed by incubation with TGF-α neutralizing antibodies and tyrphostin AG 1478, a selective and potent inhibitor of EGF-R kinase. Results: All SCC cell lines synthesized TGF-α and expressed elevated EGF-R levels compared to normal keratinocytes. Twelve of the 14 SCC biopsies contained TGF-α protein and 8 had specific EGF-R. Exogenous TGF-α or EGF significantly increased DNA synthesis in 4 of 5 SCC cell lines. TGF-α neutralizing antibodies or tyrphostin AG 1478 reduced DNA synthesis in the two SCC cell lines (FaDu and SCC9) tested. Conclusions: These results indicate that SCC cell lines and tumors usually synthesize TGF-α, have elevated levels of EGF-R, and are mitogenically stimulated by a TGF-α autocrine system. Selective inhibition of the TGF-α system by EGF-R kinase inhibitors or TGF-α neutralizing antibodies may be useful strategies for treating SCC that overexpress TGF-α and its receptor.