bluuebunny/crossref_metadata_2025 huggingface dataset:
Barrier disruption increases gene expression of cytokines and the 55 kD TNF receptor in murine skin
Authors: Wood Ladonna C. , Stalder Anna K. , Liou Amelie , Campbell Iain L. , Grunfeld Carl , Elias Peter M. , Feingold Kenneth R.
Published: April 2006

Abstract The signalling mechanisms that regulate epidermal permeability barrier homeostasis arc not known. Previous Northern blot analysis showed that both acute and chronic barrier disruption increase mRNA levels of several cytokines in murine epidermis. To further characterize the epidermal response to barrier abrogation, we used more sensitive, multi‐probe RNasc protection assays to measure the mRNA levels of additional cytokines. as well as cytokine receptors in acute and chronic models of barrier disruption. Normal mouse epidermis expressed interleukin (IL)‐lα, interferon‐γ (IFN‐γ). tumor necrosis factor‐α (TNF‐α) and IL‐6 mRNAs. Following tape‐stripping, only the mRNA levels for TNF‐α, IL‐lα, IL‐lβ and IL‐6 increased at 2.5 and 7 h, and returned toward normal levels by IX h. No mRNAs encoding TNF‐β. IL‐2, IL‐3. IL‐4 or IL‐5, were detected in the epidermis either under basal conditions or after tape‐stripping. Similarly, in a chronic model, essential fatty acid deficiency, epidermal levels of TNF‐α, IL‐lα, IL‐lβ and IL‐6 mRNAs, but not IFN‐γ mRNA. were elevated over controls; and again, mRNAs for the remaining probed cytokines were not detected. In contrast, in the dermis. only IL‐Iβ mRNA levels increased 2.5 h after tape‐stripping, and remained elevated at I8 h. mRNAs encoding the IL‐1 (p60), IFN‐γ ami IL‐6 receptors were present in epidermis, but their levels remained unchanged following either acute or chronic barrier disruption. In contrast, epidermal TNF (p55) receptor mRNA levels were increased by 87% (P<0.01) at 2.5 h, returned to control levels at 7 h and were increased by 68% (p<0.03) at 18 h after tape‐stripping. The increase at 2 h was confirmed by Northern blot analysis and was not prevented by latex occlusion performed immediately after tape‐stripping. mRNAs for the IL‐I (p80) receptor and TNF (p75) receptor were not detected in epidermis. Low levels of TNF (p55) receptor mRNA were present in the dermis. and they remained unchanged after tape‐stripping. The presence of specific receptor mRNAs in the epidermis and dermis suggests that these tissues are capable of responding in an autocrine and/or paracrine fashion to the cognate cytokines. These results suggest that epidermal cytokines produced after barrier disruption may initiate a cytokine cascade which could regulate cytokine and cytokine receptor production and/or inflammatory responses.
Propiomelanocortin (POMC) gene expression by normal skin and keloid fibroblasts in culture: modulation by cytokines
Authors: Teofoli Patrizia , Motoki Kiyohisa , Lotti Torello M. , Uitto Jouni , Mauviel Alain
Published: April 2006

Abstract Originally described as a product of the pituitary gland, propiomelanocortin (POMC) has recently been identified in other tissues, such as in human skin, where it may accumulate in response to various stimuli. Thus far, epidermal keratinocytes, as well as melanocytes and macrophages, have been shown to express POMC. This study investigated the expression of POMC mRNA in cultured dermal fibroblasts derived from either normal skin or keloids. Using Northern blot hybridization with a POMC cDNA generated by RT‐PCR of mRNA isolated from cardiac muscle, we demonstrated that dermal fibroblasts express POMC, as significant levels of mRNA were detected in unstimulated cells in culture. POMC transcript steady‐state levels were strongly reduced by transforming growth factor‐β (TGF‐β). whereas tumor necrosis factor‐α (TNF‐α) counteracted the effect of TGF‐β and exerted a stimulatory activity on POMC mRNA levels. Reduction of POMC transcript levels by TGF‐β was also observed in cultured keratinocytes. Clearly detectable levels of POMC mRNA were detected in cultured keloid‐derived fibroblasts; however, little, if any, regulation by TGF‐β was observed. These data represent the first demonstration of POMC expression by fibroblasts and down‐regulation by TGF‐β. Furthermore, our results indicate altered TGF‐β regulation of POMC gene expression in keloid‐derived fibroblasts suggesting that POMC may play a role in the pathogenesis of keloid formation.
β‐Adrenergic stimulation induces activation of protein kinase C and inositol 1,4,5‐trisphosphate increase in epidermis
Authors: Koizumi Hiroko , Tanaka Hideo , Ohkawara Akira
Published: April 2006

Abstract Epidermal keratinocytes express β2‐adrenergic receptors on the cell membrane. The binding of the agonists to the β2‐adrenergic receptors regulates activation of adenylate cyclase. This transmembrane signaling system has been regarded to be one of the important pathways for the functions of keratinocytes. We previously reported that β‐adrenergic stimulation induced a transient increase of intracellular Ca2+ in normal human epidermal keratinocytes. Thus we investigated the effects of epinephrine on another transmembrane signaling system, the phosphatidylinositol signal transduction pathway in pig epidermis. Treatment of pig pure epidermis with epinephrine resulted in a transient increase in inositol 1,4,5‐trisphosphate with a peak at 30 s. Epinephrine induced translocation of protein kinase C from cytosol to the membrane fraction. The activation of protein kinase C, translocation of protein kinase C from cytosol to the membrane fraction, was confirmed using the β‐adrenergic agonist isoproterenol. Moreover, the effect of epinephrine on the activation of protein kinase C was inhibited by preincubation with propranolol, a β‐adrenergic antagonist. The increase in inositol 1,4,5‐trisphosphate and translocation of protein kinase C by epinephrine are consistent with the view that β‐adrenergic stimulation induces turnover of inositol phospho‐lipid in pig epidermis.
Molecular analysis of different phases in human wound healing
Authors: Petri J. B. , König S. , Haupt B. , Haustein U.‐F. , Herrmann K.
Published: April 2006

Abstract Cultured granulation fibroblasts grown from punch biopsies of the same lower arm area, obtained 3, 6, 9 and 14 days after wounding, were used as a human wound healing model in comparison to quiescent fibroblasts. We investigated the expression of key extracellular matrix components at the protein level by flow cytometry and mRNA steady state levels by Northern blotting of the different fibroblasts and compared these data to the ability to migrate towards a chemotactic signal. Procollagen α1 (I), fibronectin and matrix metalloprotease‐1 synthesis was strongly upregulated at the mRNA steady state level on days 3 and 14. Tissue inhibitor of metalloprotease‐1 mRNA is only 20% down‐regulated between day 3 and 14. Chemotaxis towards conditioned medium reflects a net effect of several factors and is distinctly different from chemotaxis towards platelet‐derived growth factor, which peaks at day 3. Compared to the protein level, the enhanced expression of the corresponding PDGF receptor β chain mRNA is delayed by 3 to 6 days. PDGF receptor α shows no regulatory changes during the observation period. This data further supports the idea that functionally divergent subpopulations of fibroblasts exist during wound healing.
Isolation of a cDNA encoding a tyrosine kinase expressed in murine skin
Authors: Kawachi Y. , Nakauchi H. , Otsuka F.
Published: April 2006

Abstract Tyrosine phosphorylation is widely recognized as playing an important role in cell differentiation, proliferation and carcinogenesis. We used the polymerase chain reaction (PCR) method to identify protein tyrosine kinases that were expressed in the skin. Mixed oligonucleotide probes were used to amplify and screen neonatal murine skin mRNA for clones encoding amino acid contiguities, the conservation of which is characteristic of the protein tyrosine kinase family. When the PCR products were sequenced, a novel clone encoding protein tyrosine kinase, PTK70, was identified. A full‐length cDNA was isolated from a mouse thymus cDNA library. The nucleotide and deduced amino acid sequence showed that it featured src‐homology (SH) 2 domain, SH3 domain and kinase domain like other src family protein tyrosine kinases, but lacked the N‐terminal myristylation site and C‐terminal tyrosine residue. Although the mRNA of PTK70 was detected in various tissues ubiquitously, the degree of its expression differed among tissues. Murine skin is one in which PTK70 was expressed strongly, with its expression being much stronger in the epidermis and in the cell line derived from murine keratinocytes than in those from melanoma or fibroblast cell lines. These evidences suggest that PTK70 may be involved in proliferation or differentiation of keratinocytes in the skin.
Enkephalins modulate differentiation of normal human keratinocytes in vitro
Authors: Nissen Judith B. , Kragballe Knud
Published: April 2006

Abstract Opioid peptides are a group of neuropeptides which include enkephalins, endorphins and dynorphins. In addition to their central and peripheral antinociceptive function, opioids can modulate immune activity and cell proliferation. Previously, we have shown that enkephalins are present in macrophages infiltrating the dermal papillae in involved psoriatic skin and that the amount of enkephalin is significantly increased in involved psoriatic skin. Because enkephalins were detected close to the epidermis, we examined the effects of opioid peptides on the differentiation (transglutaminase type 1 activity and cytokeratin 10 expression) and proliferation (MTT assay) of cultured human keratinocytes. Enkephalins (methionine‐enkephalin, leucine‐cnkephalin and the synthetic DADL) inhibited cell differentiation dose‐dependently, while β‐endorphin had no effect. The opioid receptor antagonist naltrexone completely antagonized the inhibitory effect of methionine‐enkephalin and leucine‐enkephalin. but not that of DADL. Furthermore, methionine‐enkephalin had a slight inhibitory effect on the proliferation of keralinocytcs. Enkephalin was detected in unstimulated keratinocyte cultures, and naltrexone alone stimulated keratinocyte differentiation. These results indicate that enkephalins may play a role in the differentiation of epidermal keratinocytes. It remains to be determined whether the enkephalin detected in psoriatic skin are sufficient to affect epidermal differentiation in vivo.
Skin changes in “screen dermatitis” versus classical UV‐ and ionizing irradiation‐related damage — similarities and differences
Authors: Gangi S. , Johansson O.
Published: April 2006

AbstractAn increasing number of persons say that they get cutaneous problems as well as symptoms from certain internal organs, such as the central nervous system (CNS) and the heart, when being close to electric equipment. A major group of these patients are the users of video display terminals (VDTs), who claim to have subjective and objective skin‐and mucosa‐related symptoms, such as pain, itch, heat sensation, erythema, papules, and pustules. The CNS symptoms are, e.g. dizziness, tiredness, and headache. Erythema, itch, heal sensation, edema and pain are also common symptoms of sunburn (UV dermatitis). Alterations have been observed in cell populations of the skin of patients suffering from so‐called “screen dermatitis” similar to those observed in the skin damaged due to ultraviolet (UV) light or ionizing radiation. In “screen dermatitis” patients a much higher number of mast cells have been observed. It is known that UVB irradiation induces mast cell degranulation and release of TNF‐α. The high number of mast cells present in the “screen dermatitis” patients and the possible release of specific substances, such as histamine, may explain their clinical symptoms of itch, pain, edema and erythema. The most remarkable change among cutaneous cells, after exposure with the above‐mentioned irradiation sources, is the disappearance of the Langerhans' cells. This change has also been observed in “screen dermatitis” patients, again pointing to a common cellular and molecular basis. The results of this literature study demonstrate that highly similar changes exist in the skin of “screen dermatitis” patients, as regards the clinical manifestations as well as alterations in the cell populations, and in skin damaged by UV light or ionizing radiation.
Adenovirus‐mediated blockade of lymphotoxin‐β inhibits the induction of contact sensitivity in mice
Authors: Trueb Ralph M. , Brown Geri R. , Dougherty Irene , Valdez‐Silva Maria , Cruz Ponciano D.
Published: April 2006

Abstract Lymphotoxin‐β is a newly recognized member of the tumor necrosis factor ligand family. Recent studies have suggested a role for this cytokine in delayed‐type hypersensitivity responses. To determine whether lymphotoxin‐β contributes to the development of contact sensitivity, we utilized an inhibitor protein that can effectively block binding of lymphotoxin‐β to its receptor. An adenoviral vector was created that encodes for a lymphotoxin‐β inhibitor protein consisting of the extracellular domain of the lymphotoxin‐β receptor fused to IgG heavy chain. Intravenous injection of the recombinant virus into BALB/c mice yielded plasma levels of inhibitor protein >500 μg that persisted for 1 week. Mice treated in this manner were compared with control animals injected with adenovirus encoding β‐galactosidase, with respect to their ability to mount contact sensitivity responses to epicutaneously applied dinitro‐fluorobenzene. Mice transduced with the lymphotoxin‐β inhibitor prior to the induction of contact sensitivity showed significantly suppressed ear swelling responses. By contrast, mice treated with the lymphotoxin‐β inhibitor prior to the elicitation of contact sensitivity showed no change in ear swelling responses in comparison to controls. These findings indicate that lymphotoxin‐β plays an important role in the afferent phase of the contact sensitivity response.
Sodium barbital is a slow reversible inactivator of rabbit-muscle creatine kinase
Authors: Shi Feng , Zhao Tong-Jin , He Hua-Wei , Li Jie , Zeng Xian-Gang , Zhou Hai-Meng , Wu Ping
Published: April 2006

As a depressant of the central nervous system, the clinical effect of sodium barbital has been extensively studied. Here we report on sodium barbital as an inhibitor of rabbit-muscle creatine kinase (CK), which plays a significant role in energy homeostasis in the muscles. Although sodium barbital gradually inhibits the activity of CK with increased concentration, the inhibition effect can be completely reversed by dilution, indicating that the inactivation process is reversible. Detailed kinetics analysis, according to a previously presented theory, indicates that sodium barbital functions as a non complexing inhibitor, and its inhibition effect on CK is a slow reversible inactivation. In this study, a kinetic model of the substrate reaction is presented, and the microscopic rate constants for the reaction of sodium barbital with the free enzyme and the enzyme–substrate complexes are determined. Kinetic analysis reveals that sodium barbital might compete with both creatine and ATP, but mainly with creatine, to inhibit the activity of CK. The results suggest that CK might be a target for sodium barbital in vivo.Key words: creatine kinase; inactivation; kinetics; sodium barbital.
Plasma phospholipid transfer protein fused with green fluorescent protein is secreted by HepG2 cells and displays phosphatidylcholine transfer activity
Authors: Siggins Sarah , Ehnholm Christian , Jauhiainen Matti , Olkkonen Vesa M
Published: April 2006

Phospholipid transfer protein (PLTP) is a serum glycoprotein with a central role in high-density lipoprotein metabolism. We created a fusion protein in which enhanced green fluorescent protein (EGFP) was fused to the carboxyl-terminus of PLTP. Stably transfected HepG2 cells, which overexpress this fusion protein, were generated. PLTP–EGFP was translocated into the ER and fluoresced within the biosynthetic pathway, showing a marked concentration in the Golgi complex. The transfected cells secreted into the growth medium phospholipid transfer activity 7-fold higher than that of the mock-transfected controls. The medium of the PLTP–EGFP - expressing cells displayed EGFP fluorescence, demonstrating that both the PLTP and the EGFP moieties had attained a biologically active conformation. However, the specific activity of PLTP–EGFP in the medium was markedly reduced as compared with that of endogenous PLTP. This suggests that the EGFP attached to the carboxyl-terminal tail of PLTP interferes with the interaction of PLTP with its substrates or with the lipid transfer process itself. Fluorescently tagged PLTP is a useful tool for elucidating the intracellular functions of PLTP and the interaction of exogenously added PLTP with cells, and will provide a means of monitoring the distribution of exogenously added PLTP between serum lipoprotein subspecies.Key words: GFP, fusion protein, Golgi apparatus, phospholipid transfer activity, protein secretion.
Cloning and developmental characterization ofXenopus laevismembrane type-3 matrix metalloproteinase (MT3-MMP)
Authors: Hammoud Lamis , Walsh Logan A , Damjanovski Sashko
Published: April 2006

Proper extracellular matrix (ECM) remodeling, mediated by matrix metalloproteinases (MMPs), is crucial for the development and survival of multicellular organisms. Full-length Xenopus laevis membrane type-3 matrix metallo proteinase (MT3-MMP) was amplified by PCR and cloned from a stage 28 Xenopus head cDNA library. A comparison of the derived Xenopus MT3-MMP protein sequence to that of other vertebrates revealed 86% identity with human and mouse and 85% identity with chicken. The expression profile of MT3-MMP was examined during Xenopus embryogenesis: MT3-MMP transcripts were first detected at the later stages of development and were localized to dorsal and anterior structures. During metamorphosis and in the adult frog, MT3-MMP expression was restricted to specific tissues and organs. Treatment of Xenopus embryos with lithium chloride (LiCl), ultraviolet irradiation (UV), or retinoic acid (RA) revealed that MT3-MMP levels increased with LiCl-dorsalizing treatments and decreased with UV-ventralizing and RA-anterior neural truncating treatments. Overexpression of MT3-MMP through RNA injections led to dose-dependent developmental abnormalities and death. Moreover, MT3-MMP overexpression resulted in neural and head structure abnormalities, as well as truncated axes. Taken together, these results indicate that MT3-MMP expression in Xenopus is spatially and temporally restricted. Furthermore, deregulation of MT3-MMP during early embryogenesis has detrimental effects on development.Key words: Xenopus laevis, MT3-MMP, development, ECM, dorsalization, ventralization.
Cellular localization of 17 natural mutant variants of ALADIN protein in triple A syndrome – shedding light on an unexpected splice mutation
Authors: Krumbholz M , Koehler K , Huebner A
Published: April 2006

The triple A syndrome is a complex and multisystemic autosomal recessive disease with the 3 main symptoms of adrenal insufficiency, alacrima, and achalasia accompanied by neurological impairment. Mutations in the AAAS gene on chromosome 12q13 are responsible for the disorder. AAAS encodes a protein named ALADIN, which belongs to the family of WD-repeat-containing proteins and has been shown to localize to nuclear pore complexes. The function of the protein is not clear. It is supposed that ALADIN plays an important role in RNA and (or) protein trafficking between the nucleus and cytoplasm. With transfection experiments, we analyzed the cellular localization of the wild-type and 17 natural mutant variants (9 missense, 5 nonsense, 3 frameshift mutations) of ALADIN. We show that most mutations cause mislocalization of the mutant ALADIN proteins in the cytoplasm. In contrast, some variants with mutations located at the N-terminus (Q15K, L25P) and 3 artificial C-terminus mutations (Q490X, R493X, and V497X) remain at the nuclear pore. Using a patient cell line, we show that the mutation 43C>A in exon 1 does not cause a missense mutation Q15K but, rather, results in aberrant splicing.Key words: triple A syndrome, nuclear pore complex, nucleoporin, ALADIN.
A novel ribonuclease from fresh fruiting bodies of the portabella mushroom Agaricus bisporus
Authors: Wang H X , Ng T B
Published: April 2006

A 14 kDa ribonuclease with a novel N-terminal sequence was isolated from fresh fruiting bodies of the portabella mushroom. It was adsorbed on DEAE-cellulose and carboxymethyl-cellulose, and demonstrated the highest ribonucleolytic potency toward poly (A), 60% as much activity toward poly (C), 40% as much activity toward poly (U), and the least activity (7% as much) toward poly (G). It exhibited a pH optimum at pH 4.5 and a temperature optimum at 60 °C. Its activity at 100 °C was higher than that at 20 °C.Key words: ribonuclease, portabella mushroom, isolation.
Conservation ofPitx1expression during amphibian limb morphogenesis
Authors: Chang W Y , KhosrowShahian F , Wolanski M , Marshall R , McCormick W , Perry S , Crawford M J
Published: April 2006

In contrast to the pattern of limb emergence in mammals, chicks, and the newt N. viridescens, embryos such as Xenopus laevis and Eleutherodactylus coqui initiate pelvic limb buds before they develop pectoral ones. We studied the expression of Pitx1 in X. laevis and E. coqui to determine if this paired-like homeodomain transcription factor directs differentiation specifically of the hindlimb, or if it directs the second pair of limbs to form, namely the forelimbs. We also undertook to determine if embryonic expression patterns were recapitulated during the regeneration of an amputated limb bud. Pitx1 is expressed in hindlimbs in both X. laevis and E. coqui, and expression is similar in both developing and regenerating limb buds. Expression in hindlimbs is restricted to regions of proliferating mesenchyme.Key words: regeneration, Xenopus laevis, limb bud, Pitx1 protein, specification.
Quantitative imaging of basic functions in renal (patho)physiology
Authors: Kang Jung Julie , Toma Ildiko , Sipos Arnold , McCulloch Fiona , Peti-Peterdi Janos
Published: April 2006

Multiphoton fluorescence microscopy offers the advantages of deep optical sectioning of living tissue with minimal phototoxicity and high optical resolution. More importantly, dynamic processes and multiple functions of an intact organ can be visualized in real time using noninvasive methods, and quantified. These studies aimed to extend existing methods of multiphoton fluorescence imaging to directly observe and quantify basic physiological parameters of the kidney including glomerular filtration rate (GFR) and permeability, blood flow, urinary concentration/dilution, renin content and release, as well as more integrated and complex functions like the tubuloglomerular feedback (TGF)-mediated oscillations in glomerular filtration and tubular flow. Streptozotocin-induced diabetes significantly increased single-nephron GFR (SNGFR) from 32.4 ± 0.4 to 59.5 ± 2.5 nl/min and glomerular permeability to a 70-kDa fluorophore approximately eightfold. The loop diuretic furosemide 2-fold diluted and increased ∼10-fold the volume of distal tubular fluid, while also causing the release of 20% of juxtaglomerular renin content. Significantly higher speeds of individual red blood cells were measured in intraglomerular capillaries (16.7 ± 0.4 mm/s) compared with peritubular vessels (4.7 ± 0.2 mm/s). Regular periods of glomerular contraction-relaxation were observed, resulting in oscillations of filtration and tubular flow rate. Oscillations in proximal and distal tubular flow showed similar cycle times (∼45 s) to glomerular filtration, with a delay of ∼5–10 and 25–30 s, respectively. These innovative technologies provide the most complex, immediate, and dynamic portrayal of renal function, clearly depicting the components and mechanisms involved in normal physiology and pathophysiology.
Inhibition of Phosphotyrosine Phosphatase 1B Causes Resistance in BCR-ABL-Positive Leukemia Cells to the ABL Kinase Inhibitor STI571
Authors: Koyama Noriko , Koschmieder Steffen , Tyagi Sandhya , Portero-Robles Ignacio , Chromic Jörg , Myloch Silke , Nürnberger Heike , Rossmanith Tanja , Hofmann Wolf-Karsten , Hoelzer Dieter , Ottmann Oliver Gerhard
Published: April 2006

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of BCR-ABL-mediated transformation in vitro and in vivo. To investigate whether PTP1B modulates the biological effects of the abl kinase inhibitor STI571 in BCR-ABL-positive cells, we transfected Philadelphia chromosome–positive (Ph+) chronic myeloid leukemia cell-derived K562 cells with either wild-type PTP1B (K562/PTP1B), a substrate-trapping dominant-negative mutant PTP1B (K562/D181A), or empty vector (K562/mock). Cells were cultured with or without STI571 and analyzed for its effects on proliferation, differentiation, and apoptosis. In both K562/mock and K562/PTP1B cells, 0.25 to 1 μmol/L STI571 induced dose-dependent growth arrest and apoptosis, as measured by a decrease of cell proliferation and an increase of Annexin V-positive cells and/or of cells in the sub-G1 apoptotic phase. Western blot analysis showed increased protein levels of activated caspase-3 and caspase-8 and induction of poly(ADP-ribose) polymerase cleavage. Low concentrations of STI571 promoted erythroid differentiation of these cells. Conversely, K562/D181A cells displayed significantly lower PTP1B-specific tyrosine phosphatase activity and were significantly less sensitive to STI571-induced growth arrest, apoptosis, and erythroid differentiation. Pharmacologic inhibition of PTP1B activity in wild-type K562 cells, using bis(N,N-dimethylhydroxamido)hydroxooxovanadate, attenuated STI571-induced apoptosis. Lastly, comparison of the STI571-sensitive Ph+ acute lymphoblastic leukemia cell line SupB15 with a STI571-resistant subline revealed significantly decreased PTP1B activity and enhanced BCR-ABL phosphorylation in the STI571-resistant SupB15 cells. In conclusion, functional PTP1B is involved in STI571-induced growth and cell cycle arrest, apoptosis, and differentiation, and attenuation of PTP1B function may contribute to resistance towards STI571.
Altered Nuclear Receptor Corepressor Expression Attenuates Vitamin D Receptor Signaling in Breast Cancer Cells
Authors: Banwell Claire M. , MacCartney Donia P. , Guy Michelle , Miles Alice E. , Uskokovic Milan R. , Mansi Janine , Stewart Paul M. , O'Neill Laura P. , Turner Bryan M. , Colston Kay W. , Campbell Moray J.
Published: April 2006

Purpose: We hypothesized that deregulated corepressor actions, with associated histone deacetylation activity, epigenetically suppressed vitamin D receptor (VDR) responsiveness and drives resistance towards 1α,25-dihydroxyvitamin D3.

Experimental Design: Profiling, transcriptional, and proliferation assays were undertaken in 1α,25(OH)2D3-sensitive MCF-12A nonmalignant breast epithelial cells, a panel of breast cancer cell lines, and a cohort of primary breast cancer tumors (n = 21).

Results: Elevated NCoR1 mRNA levels correlated with suppressed regulation of VDR target genes and the ability of cells to undergo arrest in G1 of the cell cycle. A similar increased ratio of corepressor mRNA to VDR occurred in matched primary tumor and normal cells, noticeably in estrogen receptor α–negative (n = 7) tumors. 1α,25(OH)2D3 resistance in cancer cell lines was targeted by cotreatments with either 1α,25(OH)2D3 or a metabolically stable analogue (RO-26-2198) in combination with either trichostatin A (TSA; histone deacetylation inhibitor) or 5-aza-2′-deoxycytidine (DNA methyltransferase inhibitor). Combinations of vitamin D3 compounds with TSA restored VDR antiproliferative signaling (target gene regulation, cell cycle arrest, and antiproliferative effects in liquid culture) to levels which were indistinguishable from MCF-12A cells.

Conclusions: Increased NCoR1 mRNA is a novel molecular lesion in breast cancer cells, which acts to suppress responsiveness of VDR target genes, resulting in 1α,25(OH)2D3 resistance and seems to be particularly associated with estrogen receptor negativity. This lesion provides a novel molecular diagnostic and can be targeted by combinations of vitamin D3 compounds and low doses of TSA.
A Two-Gene Expression Ratio of Homeobox 13 and Interleukin-17B Receptor for Prediction of Recurrence and Survival in Women Receiving Adjuvant Tamoxifen
Authors: Goetz Matthew P. , Suman Vera J. , Ingle James N. , Nibbe Andrea M. , Visscher Dan W. , Reynolds Carol A. , Lingle Wilma L. , Erlander Mark , Ma Xiao-Jun , Sgroi Dennis C. , Perez Edith A. , Couch Fergus J.
Published: April 2006

Purpose: In the adjuvant treatment of estrogen receptor (ER)–positive breast cancer, additional markers are needed to identify women at high risk for recurrence.

Experimental Design: We examined the association between the ratio of the homeobox 13 (HOXB13) to interleukin-17B receptor (IL-17BR) expression and the clinical outcomes of relapse and survival in women with ER-positive breast cancer enrolled onto a North Central Cancer Treatment Group adjuvant tamoxifen trial (NCCTG 89-30-52).

Results: Tumor blocks were obtained from 211 of 256 eligible patients, and quantitative reverse transcription-PCR profiles for HOXB13 and IL-17BR were obtained from 206 patients. The cut point for the two-gene log 2(expression ratio) that best discriminated clinical outcome (recurrence and survival) was selected and identified women with significantly worse relapse-free survival (RFS), disease-free survival (DFS), and overall survival (OS), independent of standard prognostic markers. The cut point differed as a function of nodal status [node negative (59th percentile) versus node positive (90th percentile)]. In the node-positive cohort (n = 86), the HOXB13/IL-17BR ratio was not associated with relapse or survival. In contrast, in the node-negative cohort (n = 130), a high HOXB13/IL-17BR ratio was associated with significantly worse RFS [hazard ratio (HR), 1.98; P = 0.031], DFS (HR, 2.03; P = 0.015), and OS (HR, 2.4; P = 0.014), independent of standard prognostic markers.

Conclusion: A high HOXB13/IL-17BR expression ratio is associated with increased relapse and death in patients with resected node-negative, ER-positive breast cancer treated with tamoxifen and may identify patients in whom alternative therapies should be studied.
Evaluation of Adverse Events Experienced by Older Patients Participating in Studies of Molecularly Targeted Agents Alone or in Combination
Authors: Townsley Carol A. , Pond Gregory R. , Oza Amit M. , Hirte Hal W. , Winquist Eric , Goss Glenwood , Degendorfer Pamela , Moore Malcolm J. , Siu Lillian L.
Published: April 2006

Background: The tolerability of molecularly targeted agents in older patients has not been specifically examined. Adverse event data from clinical trials in the Princess Margaret Hospital Phase II Consortium database were analyzed to address this question.

Methods: The Consortium database collects trial information on all patients treated with either a molecularly targeted agent alone or in combination since 2001. The frequency of adverse events was determined and analyzed by two different age groups, <65 years and ≥65 years. Toxicity indices (TI) and frequencies of dose-limiting toxicities (DLT), based on adverse events of all causalities (TIALL and DLTALL), and on adverse events that were at least possibly related to the molecularly targeted agent (TIMTA and DLTMTA), were calculated for both age groups.

Results: Four hundred and one patients who received 1,252 treatment cycles were analyzed from 19 different studies. Baseline performance status was similar between both age groups, but fewer older patients have had multiple prior regimens of chemotherapy or prior radiation therapy. A comparison of the proportions of younger and older patients experiencing DLTALL and DLTMTA showed similar results. The TIMTA values were comparable between the two age groups in both single agent (3.25 versus 3.00, for <65 versus ≥65 years) and multi-agent (3.65 versus 3.00, for <65 versus ≥65 years) trials.

Conclusions: Older patients seem to tolerate molecularly targeted therapies either alone or in combination with chemotherapy as well as younger patients. Age alone should not be a barrier in the administration of targeted agents.
Differentially Regulated Micro-RNAs and Actively Translated Messenger RNA Transcripts by Tumor Suppressor p53 in Colon Cancer
Authors: Xi Yaguang , Shalgi Reut , Fodstad Oystein , Pilpel Yitzhak , Ju Jingfang
Published: April 2006

Purpose: The aim of this study was to investigate the role of p53 in regulating micro-RNA (miRNA) expression due to its function as a transcription factor. In addition, p53 may also affect other cellular mRNA gene expression at the translational level either via its mediated miRNAs or due to its RNA-binding function.

Experimental Design: The possible interaction between p53 and miRNAs in regulating gene expression was investigated using human colon cancer HCT-116 (wt-p53) and HCT-116 (null-p53) cell lines. The effect of p53 on the expression of miRNAs was investigated using miRNA expression array and real-time quantitative reverse transcription-PCR analysis.

Results: Our investigation indicated that the expression levels of a number of miRNAs were affected by wt-p53. Down-regulation of wt-p53 via small interfering RNA abolished the effect of wt-p53 in regulating miRNAs in HCT-116 (wt-p53) cells. Global sequence analysis revealed that over 46% of the 326 miRNA putative promoters contain potential p53-binding sites, suggesting that some of these miRNAs were potentially regulated directly by wt-p53. In addition, the expression levels of steady-state total mRNAs and actively translated mRNA transcripts were quantified by high-density microarray gene expression analysis. The results indicated that nearly 200 cellular mRNA transcripts were regulated at the posttranscriptional level, and sequence analysis revealed that some of these mRNAs may be potential targets of miRNAs, including translation initiation factor eIF-5A, eIF-4A, and protein phosphatase 1.

Conclusion: To the best of our knowledge, this is the first report demonstrating that wt-p53 and miRNAs interact in influencing gene expression and providing insights of how p53 regulates genes at multiple levels via unique mechanisms.
Delineation, Functional Validation, and Bioinformatic Evaluation of Gene Expression in Thyroid Follicular Carcinomas with the PAX8-PPARG Translocation
Authors: Giordano Thomas J. , Au Amy Y.M. , Kuick Rork , Thomas Dafydd G. , Rhodes Daniel R. , Wilhelm Kenneth G. , Vinco Michelle , Misek David E. , Sanders Donita , Zhu Zhaowen , Ciampi Raffaele , Hanash Samir , Chinnaiyan Arul , Clifton-Bligh Roderick J. , Robinson Bruce G. , Nikiforov Yuri E. , Koenig Ronald J.
Published: April 2006

A subset of follicular thyroid carcinomas contains a balanced translocation, t(2;3)(q13;p25), that results in fusion of the paired box gene 8 (PAX8) and peroxisome proliferator-activated receptor γ (PPARG) genes with concomitant expression of a PAX8-PPARγ fusion protein, PPFP. PPFP is thought to contribute to neoplasia through a mechanism in which it acts as a dominant-negative inhibitor of wild-type PPARγ. To better understand this type of follicular carcinoma, we generated global gene expression profiles using DNA microarrays of a cohort of follicular carcinomas along with other common thyroid tumors and used the data to derive a gene expression profile characteristic of PPFP-positive tumors. Transient transfection assays using promoters of four genes whose expression was highly associated with the translocation showed that each can be activated by PPFP. PPFP had unique transcriptional activities when compared with PAX8 or PPARγ, although it had the potential to function in ways qualitatively similar to PAX8 or PPARγ depending on the promoter and cellular environment. Bioinformatics analyses revealed that genes with increased expression in PPFP-positive follicular carcinomas include known PPAR target genes; genes involved in fatty acid, amino acid, and carbohydrate metabolism; micro-RNA target genes; and genes on chromosome 3p. These results have implications for the neoplastic mechanism of these follicular carcinomas.
Performance of Immuno–Positron Emission Tomography with Zirconium-89-Labeled Chimeric Monoclonal Antibody U36 in the Detection of Lymph Node Metastases in Head and Neck Cancer Patients
Authors: Börjesson Pontus K.E. , Jauw Yvonne W.S. , Boellaard Ronald , de Bree Remco , Comans Emile F.I. , Roos Jan C. , Castelijns Jonas A. , Vosjan Maria J.W.D. , Kummer J. Alain , Leemans C. René , Lammertsma Adriaan A. , van Dongen Guus A.M.S.
Published: April 2006

Purpose: Immuno–positron emission tomography (PET), the combination of PET with monoclonal antibodies (mAb), is an attractive option to improve tumor detection and to guide mAb-based therapy. The long-lived positron emitter zirconium-89 (89Zr) has ideal physical characteristics for immuno-PET with intact mAbs but has never been used in a clinical setting. In the present feasibility study, we aimed to evaluate the diagnostic imaging performance of immuno-PET with 89Zr-labeled-chimeric mAb (cmAb) U36 in patients with squamous cell carcinoma of the head and neck (HNSCC), who were at high risk of having neck lymph node metastases.

Experimental Design: Twenty HNSCC patients, scheduled to undergo neck dissection with or without resection of the primary tumor, received 75 MBq 89Zr coupled to the anti-CD44v6 cmAb U36 (10 mg). All patients were examined by computed tomography (CT) and/or magnetic resonance imaging (MRI) and immuno-PET before surgery. Six patients also underwent PET with 18F-fluoro-2-deoxy-d-glucose. Immuno-PET scans were acquired up to 144 hours after injection. Diagnostic findings were recorded per neck side (left or right) as well as per lymph node level (six levels per side), and compared with histopathologic outcome. For this purpose, the CT/MRI scores were combined and the best of both scores was used for analysis.

Results: Immuno-PET detected all primary tumors (n = 17) as well as lymph node metastases in 18 of 25 positive levels (sensitivity 72%) and in 11 of 15 positive sides (sensitivity 73%). Interpretation of immuno-PET was correct in 112 of 121 operated levels (accuracy 93%) and in 19 of 25 operated sides (accuracy 76%). For CT/MRI, sensitivities of 60% and 73% and accuracies of 90% and 80% were found per level and side, respectively. In the six patients with seven tumor-involved neck levels and sides, immuno-PET and 18F-fluoro-2-deoxy-d-glucose PET gave comparable diagnostic results.

Conclusion: In this study, immuno-PET with 89Zr-cmAb U36 performed at least as good as CT/MRI for detection of HNSCC lymph node metastases.
Population Pharmacokinetics of Troxacitabine, a Novel Dioxolane Nucleoside Analogue
Authors: Lee Carlton K. K. , Rowinsky Eric K. , Li Jing , Giles Francis , Moore Malcolm J. , Hidalgo Manuel , Capparelli Edmund , Jolivet Jacques , Baker Sharyn D.
Published: April 2006

Purpose: To develop and validate a population pharmacokinetic model for troxacitabine, a novel l-nucleoside analogue, administered by short infusion; to characterize clinical covariates that influence pharmacokinetic variability; and to design a dosage rate for continuous infusion administration to achieve low micromolar concentrations, which may be more efficacious than shorter infusions.

Experimental Design: Plasma samples from 111 cancer patients receiving troxacitabine (0.12-12.5 mg/m2) as a 30-minute infusion in phase I trials were used to develop the model with NONMEM. Clinical covariates evaluated included creatinine clearance, body surface area, age, and sex. From the model, a troxacitabine dosage rate of 2.0 to 3.0 mg/m2/d was expected to achieve a target concentration of 0.1 μmol/L; plasma samples were obtained during the infusion from eight patients receiving troxacitabine as a 3-day infusion.

Results: Troxacitabine pharmacokinetics were characterized by a three-compartment linear model. The mean value for systemic clearance [interindividual variability (CV%)] from the covariate-free model was 9.1 L/h (28%). Creatinine clearance and body surface area accounted for 36% of intersubject variation in clearance. Troxacitabine 2.0 mg/m2/d (n = 3) and 3.0 mg/m2/d (n = 5) for 3 days produced mean ± SD end of infusion concentrations of 0.12 ± 0.03 and 0.15 ± 0.03 μmol/L, respectively.

Conclusions: Renal function and body surface area were identified as sources of troxacitabine pharmacokinetic variability. The population pharmacokinetic model model–derived dosage rates for continuous infusion administration successfully achieved predetermined target plasma concentrations. The present model may be used to optimize treatment with troxacitabine by developing a dosing strategy based on both renal function and body size.
Identification of Gains on 1q and Epidermal Growth Factor Receptor Overexpression as Independent Prognostic Markers in Intracranial Ependymoma
Authors: Mendrzyk Frank , Korshunov Andrey , Benner Axel , Toedt Grischa , Pfister Stefan , Radlwimmer Bernhard , Lichter Peter
Published: April 2006

Purpose: Pathogenesis of ependymomas is still poorly understood and molecular markers for risk-adapted patient stratification are not available. Our aim was to screen for novel genomic imbalances and prognostic markers in ependymal tumors.

Experimental Design: We analyzed 68 sporadic tumors by matrix-based comparative genomic hybridization using DNA microarrays containing >6,400 genomic DNA fragments. Novel recurrent genomic gains were validated by fluorescence in situ hybridization using a tissue microarray consisting of 170 intracranial ependymomas. Candidate genes were also tested for mRNA expression by quantitative real-time PCR, and protein expression was determined by immunohistochemistry on the tissue microarray.

Results: Chromosomal gain of 1q correlated with pediatric patients (P = 0.004), intracranial ependymomas (P = 0.05), and tumors of grade III (P = 0.002). Gain of 1q21.1-32.1 was associated with tumor recurrence in intracranial ependymomas (P < 0.001). Furthermore, gain of 1q25 as determined by fluorescence in situ hybridization represented an independent prognostic marker for either recurrence-free survival (P < 0.001) or overall survival (P = 0.003). Recurrent gains at 5p15.33 covering hTERT were validated by immunohistochemistry, and elevated protein levels correlated with adverse prognosis (P = 0.01). In addition to frequent gains and high-level amplification of epidermal growth factor receptor (EGFR) at 7p11.2, immunohistochemistry revealed protein overexpression to be correlated with poor prognosis (P = 0.002). EGFR protein status subdivides intracranial grade II ependymomas into two different risk groups (P = 0.03) as shown by multivariate analysis.

Conclusions: Thus, the states of 1q25 and EGFR represent independent prognostic markers for intracranial ependymomas to identify patient subgroups with different risk profiles in further clinical investigations. Moreover, EGFR might serve as therapeutic target for more specific chemotherapy applications.
Targeting the Mitogen-Activated Protein Kinase Pathway in the Treatment of Malignant Melanoma
Authors: Panka David J. , Atkins Michael B. , Mier James W.
Published: April 2006

The mitogen-activated protein kinase (MAPK; i.e., Ras–Raf–Erk) pathway is an attractive target for therapeutic intervention in melanoma due to its integral role in the regulation of proliferation, invasiveness, and survival and the recent availability of pharmaceutical agents that inhibit the various kinases and GTPases that comprise the pathway. Genetic studies have identified activating mutations in either B-raf or N-ras in most cutaneous melanomas. Other studies have delineated the contribution of autocrine growth factors (e.g., hepatocyte growth factor and fibroblast growth factor) to MAPK activation in melanoma. Still, others have emphasized the consequences of the down-modulation of endogenous raf inhibitors, such as Sprouty family members (e.g., SPRY2) and raf-1 kinase inhibitory protein, in the regulation of the pathway. The diversity of molecular mechanisms used by melanoma cells to ensure the activity of the MAPK pathway attests to its importance in the evolution of the disease and the likelihood that inhibitors of the pathway may prove to be highly effective in melanoma treatment. MAPK inhibition has been shown to result in the dephosphorylation of the proapoptotic Bcl-2 family members Bad and Bim. This process in turn leads to caspase activation and, ultimately, the demise of melanoma cells through the induction of apoptosis. Several recent studies have identified non–mitogen-activated protein/extracellular signal-regulated kinase kinase–binding partners of raf and suggested that the prosurvival effects of raf and the lethality of raf inhibition are mediated through these alternative targets, independent of the MAPK pathway. Other studies have suggested that endothelial cells are the primary targets of raf inhibitors in vivo and that the antitumor effect of these agents are largely attributable to angiogenesis inhibition. This article reviews the genetic and biochemical factors contributing to MAPK activation in melanoma, the mechanisms by which inhibition of the pathway might prove deleterious to tumor cells, and the potential of MAPK inhibitors in the treatment of the disease.
Immune Monitoring of T-Cell Responses in Cancer Vaccine Development
Authors: Keilholz Ulrich , Martus Peter , Scheibenbogen Carmen
Published: April 2006

Monitoring cellular immune responses is one prerequisite for rational development of cancer vaccines. The primary objective of immune monitoring is to determine the efficacy of a vaccine to induce or augment a specific T-cell response. Further questions relate to the prevalence and functional relevance of spontaneous tumor-directed immune responses, the functional characteristics of T-cell responses, and, finally and most importantly, the relationship between immune monitoring assay results and clinical end points. The issue of T-cell monitoring has become more complex as different types and generations of assays have been adopted during the past decade and both standardization and validation of assays have often been insufficient. Because the development of assays parallel the clinical development of cancer vaccines, technical advances have been achieved simultaneously with broadening understanding of cancer immunity. Suitable animal models for immune monitoring are, however, lacking, because preclinical vaccine development in rodents does not allow serial immune monitoring of the peripheral blood, as is commonly used in patients. The current situation is characterized by a lack of universal standards for T-cell assessment, uncertainty about the association between immune monitoring assay results and clinical antitumor end points, and lack of knowledge regarding the contribution of different aspects of T-cell function to clinical efficacy. It is acknowledged that T-cell monitoring will have to be validated in large trials with clinically effective vaccines, but this necessity should not discourage the current application of novel assays within clinical trials of all stages.