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Background: NJLCG1402 was a phase I/II trial investigating biweekly nanoparticle albumin-bound paclitaxel (nab-PTX) in patients with advanced non-small cell lung cancer (NSCLC)., Methods: The study included patients aged ≥20 years with previously treated NSCLC. Nab-PTX (100-150 mg/m 2 ) was administered biweekly in a 28-day cycle. The phase I portion was performed to determine the recommended phase II dose of nab-PTX. In the phase II portion, the primary endpoint was the objective response rate. Secondary endpoints were disease control rate, progression-free survival, overall survival, and safety., Results: A total of 15 patients received biweekly nab-PTX (100-150 mg/m 2 ) and 12 patients in phase II were treated with 150 mg/m 2 . In the phase I portion, 150 mg/m 2 was determined as the recommended dose. Among those treated with 150 mg/m 2 , the objective response rate was 22%, and the median progression-free and overall survival was 3.6 and 11.2 months, respectively. Adverse events grade ≥3 were observed in 39% of patients., Conclusions: Biweekly nab-PTX monotherapy was well tolerated and exhibited favorable antitumor activity in patients with previously treated NSCLC., (© 2021 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Background: A variant of c.14429G>A (p.Arg4810Lys, rs112735431) in the ring finger protein 213 gene (RNF213; NM&#95;001256071.2) has been recently identified as a risk allele for pulmonary arterial hypertension (PAH). PAH can be added as a new member of RNF213-associated vascular diseases, which include Moyamoya disease and peripheral pulmonary stenosis. Our aim was to identify the clinical features and outcomes of PAH patients with this variant., Methods: Whole-exome sequencing was performed in 139 idiopathic (or possibly heritable) PAH patients., Results: The RNF213 p.Arg4810Lys variant was identified in a heterozygous state in 11 patients (7.9%). Time-course changes in hemodynamics after combination therapy in the patients with the RNF213 p.Arg4810Lys variant were significantly poorer compared with those carrying the bone morphogenic protein receptor type 2 (BMPR2) mutation (n = 36) (comparison of changes in mean pulmonary arterial pressure, p = 0.007). The event-free rate of death or lung transplantation was significantly poorer in RNF213 p.Arg4810Lys variant carriers than in BMPR2 mutation carriers (5-year event-free rate since the introduction of prostaglandin I 2 infusion, 0% vs 93%, respectively; p < 0.001)., Conclusions: Idiopathic PAH patients with the RNF213 p.Arg4810Lys variant are associated with poor clinical outcomes even in recent times. Earlier consideration of lung transplantation might be required for RNF213 p.Arg4810Lys variant carriers who are developing PAH. Documentation of the RNF213 p.Arg4810Lys variant, as well as already known pathogenic genes, such as BMPR2, can provide clinically relevant information for therapeutic strategies, leading to a personalized approach for the treatment of PAH., (Copyright © 2019 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

A substantial proportion of patients with pulmonary arterial hypertension (PAH) have mutations in the Bone Morphogenetic Protein Receptor type-2 (BMPR2) gene. PAH due to BMPR2 mutations is inherited as an autosomal dominant trait with several unique features, including a wide variety of mutations, reduced penetrance, a skewed gender ratio, variable expressivity and genetic anticipation. To address the genetic background of these unique features of BMPR2 mutation, we conducted a systematic analysis of 15 PAH families with BMPR2 mutation. The exonic protein coding sequence of BMPR2 was amplified by polymerase chain reaction and the products were sequenced directly to detect point mutations in BMPR2. Parental identification was carried out to confirm the parental relationship using multiplex 15 loci analysis. Combining mutation detection in family members with parental identification, we described three cases of de novo mutation in the BMPR2 gene by different modes in a PAH family. These de novo mutations may account for the wide variety of mutations in BMPR2. Taken together with the juvenile onset of the disease, there is possibly some balance of de novo mutations and untransmittable mutations which keeps the frequency of PAH low in the general population., (© 2015 John Wiley & Sons Ltd/University College London.)

The antisense RNA ArrS is complementary to a sequence in the 5' untranslated region of the gadE T3 mRNA, the largest transcript of gadE, which encodes a transcriptional activator of the glutamate-dependent acid resistance system in Escherichia coli. Expression of arrS is strongly induced during the stationary growth phase, particularly under acidic conditions, and transcription is dependent on σ(S) and GadE. The aim of the present study was to clarify the role of ArrS in controlling gadE expression by overexpressing arrS in E. coli. The results showed a marked increase in the survival of arrS-overexpressing cells at 2 h after a shift to pH 2.5. This was accompanied by increased expression of gadA, gadBC and gadE. The level of gadE T3 mRNA decreased markedly in response to arrS overexpression, and was accompanied by a marked increase in gadE mRNA T2. T2 mRNA had a monophosphorylated 5' terminus, which is usually found in cleaved mRNAs, and no T2 mRNA was observed in an RNase III-deficient cell strain. In addition, T2 mRNA was not generated by a P3-deleted gadE-luc translational fusion. These results suggest strongly that T2 mRNA is generated via the processing of T3 mRNA. Moreover, the T2 mRNA, which was abundant in arrS-overexpressing cells, was more stable than T3 mRNA in non-overexpressing cells. These results suggest that overexpression of ArrS positively regulates gadE expression in a post-transcriptional manner.

The presence of genetic rearrangements of bone morphogenetic protein type 2 receptor (BMPR2) was identified in pulmonary arterial hypertension (PAH) patients as the deletion or duplication of one or more exons of the gene. We recently investigated the deletion break points in exonic deletions of BMPR2 in two Japanese familial cases with PAH, and found that these were Alu-mediated via either non-allelic homologous recombination or non-homologous recombination. We herein report the third case of exonic deletion, which was in a 25-year-old female PAH patient with a deletion of BMPR2 exon 3. The break point in this case was not located in an Alu sequence. The 5'- and 3'-break point maps between the inverted Alu sequences in intron 2 and in exon 3, respectively, resulted in a 759-bp deletion. This novel exonic deletion in this PAH case may be a unique and non-recurrent rearrangement, and appears to be of a different size from that in other patients.

Purpose: The purpose of this study was to undertake thorough genetic analysis of the bone morphogenetic protein type 2 receptor (BMPR2) gene in patients with pulmonary arterial hypertension., Methods: We conducted a systematic analysis for larger gene rearrangements together with conventional mutation analysis in 152 pulmonary arterial hypertension patients including 43 patients diagnosed as having idiopathic pulmonary arterial hypertension and 10 diagnosed as having familial pulmonary arterial hypertension., Results: Analysis of the BMPR2 gene revealed each of the four kinds of nonsense and frameshift mutations, one missense mutation, one splice-site mutation, and two types of exonic deletion. For cases in which exons 1-3 were deleted, the 5' and 3' break points were located in the AluY repeat sequences in the 5' side of the adjacent NOP58 gene and in the AluY repeat sequences in intron 3, suggesting an AluY-mediated nonallelic homologous recombination as the mechanism responsible for the deletion. For the case in which exon 10 was deleted, nonhomologous recombination took place between the AluSx site in intron 9 and a unique sequence in intron 10., Conclusion: Exonic deletions of BMPR2 account for at least part of BMPR2 mutations associated with heritable pulmonary arterial hypertension in Japan, as previously reported in other populations. One of our cases was mediated via Alu-mediated nonallelic homologous recombination and another was mediated via nonhomologous recombination.

6H57, a 69-nucleotide-long small RNA, was isolated in shotgun cloning using an RNA sample derived from early stationary-phase cells. The 6H57 gene is located in a 798-bp intergenic region between two acid resistance-related genes, hdeD and gadE, and is encoded on the strand opposite these flanking genes. In this study, we carried out stringent Northern blotting to determine target mRNAs of 6H57. A band approximately 1300 nucleotides in length was detected using a probe containing a partial sequence of 6H57 and was confirmed to be the gadE mRNA T3, which has a 566-nucleotide-long 5' untranslated region. These results show that 6H57 is an antisense RNA of gadE mRNA T3 and can base pair with a -380 to -312 region of the translation initiation site of gadE. We analyzed the transcription of 6H57 and showed that 6H57 transcription is dependent on GadE in the early stationary phase. Furthermore, 6H57 is induced in the exponential growth phase by an acid stimulus of pH 5.5. A 189-bp DNA fragment containing the upstream region of the 6H57 gene showed clear promoter activities in these culture conditions. These results suggest that 6H57 plays several roles in acid resistance, and we renamed it acid resistance-related small RNA., (© 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.)

Background: Among many neurotransmitter systems in the central nervous system, the cholinergic system has been shown to contribute to propofol's sedative/anesthetic effects, because it has been shown that cholinesterase inhibitor reverses the level of propofol-induced unconsciousness in humans. It has been reported that intraperitoneal injection of propofol induced sedative/anesthetic actions and decreased the release of acetylcholine (Ach) from the rat cortex. However, the sites of action of propofol in the cholinergic pathway and its related pathways remain unresolved. We studied whether microinjection of propofol into the nuclei in the cholinergic pathway and its related pathways may induce sedation and decrease Ach from the cortex., Methods: Thirty-seven male Wistar rats weighing 270 to 320 g were used. Almost 5 days before the experiments, 23 rats anesthetized with pentobarbital (50 mg/kg) were outfitted with an electroencephalogram (EEG) socket, a microdialysis cannula in the cortex, and an intraperitoneal tube or a microinjection tube into the basal forebrain (BF), the perifornical area (Pef), or the striatum. The Ach effluxes in the somatosensory cortex were detected using in vivo intracerebral microdialysis in freely moving rats. Once basal levels of Ach were stabilized, samples were collected every 20 minutes and measured by high-performance liquid chromatography. In the intraperitoneal group, propofol was cumulatively administered (10, 30, and 100 mg/kg) into the peritoneal cavity. In the microinjection groups, propofol (40 ng in 0.2 microL) was administered into the BF, the Pef, or the striatum (control), and the cortical changes in Ach efflux and EEG were observed for 2 hours. In another 14 rats, the sedative/anesthetic score was obtained after intraperitoneal, Pef, or striatal injection of propofol. The placement of the tip of the microdialysis probe and the microinjection tube was confirmed by histological examination., Results: Intraperitoneal injection of propofol dose-dependently decreased the Ach efflux and induced light sedative to moderate anesthetic states. Loss of righting reflex was observed with significant increases in the relative alpha-power band at 100 mg/kg propofol. Microinjection of propofol into the BF significantly decreased the cortical Ach efflux to -40.2% + or - 19.9% at 40 to 60 minutes. However, there was no difference in the total Ach efflux for 2 hours between BF and control groups. In contrast, microinjection of propofol into the Pef immediately decreased the Ach efflux at 0 to 20 min and maximally to -59.3 + or - 20.4 at 100 to 120 minutes. The total Ach efflux in the Pef microinjection group was significantly less than that in the control group. The same dose of propofol injected into the Pef induced light to deep sedation. There was no significant change in the relative EEG power band between BF or Pef and control groups., Conclusion: The nuclei in the Pef are, at least in part, responsible for the sedative action of propofol in rats.

Objective: Dysfunction of the anterior spinal arteries (ASAs) may induce paresis or paraplegia after thoracoabdominal aortic aneurysm or spine surgery. However, there have been no reports of the effects of CO2 and pH on ASAs. Information on these effects on ASAs might contribute to the perioperative management or critical care of spinal cord function. Thus, we investigated the effects of CO2 and pH on the vasomotor tone of ASAs and the third branch of the middle cerebral artery (bMCA)., Design: Prospective study of the effects of CO2 and pH on vasomotor response of porcine ASA and bMCA in vitro., Setting: University laboratories., Subjects: Porcine heads and spinal cords obtained from a slaughterhouse., Intervention: ASAs and bMCAs were isolated, and changes in the intraluminal region of these pressurized arteries ( approximately 80 mm Hg) were observed for 30 minutes after perfusion with a solution saturated with various concentrations of CO2 and pH., Measurements and Main Results: Respiratory acidosis (pH/Pco2 approximately 7.10-7.15/ approximately 60-80 mm Hg) constricted the ASAs, followed by a partial but gradual decrease in tone, whereas the bMCAs were exclusively dilated. The respiratory alkalosis (pH/Pco2 approximately 7.60/ approximately 20 mm Hg) did not influence ASA tone. Vasoconstriction of the ASAs induced by respiratory acidosis was abolished by removal of the endothelium, but not by N-nitro-L-arginine (1 microM). Respiratory acidosis dilated the ASAs in all preparations treated with ONO-3708 (1 microM), a specific thromboxane A2 receptor antagonist, and OKY-046 (1 microM), a specific thromboxane synthase inhibitor. Metabolic acidosis (pH/Pco2 approximately 7.10/ approximately 40 mm Hg) caused dilation of both bMCAs and ASAs, which was abolished by glibenclamide (1 microM)., Conclusions: CO2-induced endothelium-dependent constriction in porcine ASAs through releasing thromboxane A2-like substance(s). Thus, hypercarbia might not be favorable for the perioperative or critical care management of spinal cord function during thoracoabdominal aortic aneurysm and spine surgery.

Object: To establish a new method for the diagnosis of central nervous system diseases, the authors visualized the cerebral cisterns and ventricles via a percutaneous lumbosacral route by using newly developed fine, flexible fiberscopes., Methods: Fine, flexible fiberscopes, 0.9 and 1.4 mm in diameter, were introduced up to the cerebral cisterns and ventricles through a percutaneous lumbosacral route in awake patients with chronic headache and/or neck pain or those undergoing spinal surgery and in whom MR imaging did not disclose any particular abnormalities in the brain. A lumbosacral subarachnoid puncture was made with a modified method of a continuous epidural block., Results: In 25 of 31 patients tested, the cerebellomedullary and/or pontine/interpeduncular cisterns were easily and safely reached, and the brainstem structures were visualized. Advancement of the fiberscope beyond the spinal level was abandoned in 6 patients with adhesive spinal arachnoiditis, because the fiberscopes encountered resistance seemingly caused by arachnoid adhesions. Further advancement of the fiberscopes up to the fourth and third ventricles was successfully achieved in 2 patients. A number of arachnoid filaments were found in the cerebellomedullary cistern in 4 patients: 2 with chronic spinal arachnoiditis, 1 with a spinal arachnoid cyst, and 1 with posttraumatic pain syndrome. None of the patients reported pain or any major complication except a postspinal headache and light fever, which were encountered in 4 and 1 patient, respectively., Conclusions: The approach to the supraspinal structures via the lumbosacral route by using a fine, flexible fiberscope may provide a new, minimally invasive, and safe way to observe the cerebral cisterns and/or brainstem regions.

Heavy metal pollution of soil causes biological problems, such as mutagenicity to living organisms, including human beings. However, few methods have been developed to assess metal mutagenicity in soil. To avoid metal mutagenicity, an adequate bio-monitoring method is required. In the present study, to determine if the analysis of oxidative DNA damage generated in the earthworm is a useful bio-monitoring method for soil mutagenicity, the accumulation of 8-hydroxydeoxyguanosine (8-OH-dG), a major form of oxidative DNA damage, in Eisenia fetida (Savigny, 1826) treated with cadmium chloride (CdCl2) or nickel chloride (NiCl2) was analyzed. E. fetida was treated with Cd (10 or 200 microg/g soil) or Ni (10 or 200 microg/g soil) for 1, 2, and 3 weeks or 3 months. After metal exposure, the metal concentration in E. fetida was analyzed by atomic absorption spectrometry and the 8-OH-dG accumulated in E. fetida was analyzed by HPLC analyses and immunohistochemistry. Atomic absorption spectrometry revealed that Cd, but not Ni, accumulated within E. fetida. The 8-OH-dG levels in the DNA of E. fetida treated with Cd for 3 months were significantly higher than those in control E. fetida. Moreover, immunohistochemical analyses revealed that positive signals for 8-OH-dG accumulation in seminal vesicles were detected only in E. fetida treated with 10 microg of Cd for 3 months. Although some points remain unresolved, a bio-monitoring system analyzing the DNA damage generated in the earthworm might be useful for the assessment of the mutagenicity of soil contaminated with various heavy metals, such as Cd.

Creatine kinase (CK) is a member of a group of phosphoryl transfer enzymes called phosphagen kinases that play a key role in cellular energy transactions in animals. Three CK isoform gene families are known-cytoplasmic CK (CK), flagellar CK (fCK), and mitochondrial CK (MiCK). Each of the isoforms has a unique gene structure (intron/exon organization). A broad array of other phosphagen kinases is present in animals. Some of these enzymes are found only in annelids and closely related groups including glyocyamine kinase (GK), lombricine kinase (LK), taurocyamine kinase (TK), and a unique arginine kinase (AK) restricted to annelids. Phylogenetic analyses of these annelid phosphagen kinases indicate that they appear to have evolved from a CK-like ancestor. To gain a greater understanding of the relationship of the CK isoforms to the annelid enzymes, we have determined the intron/exon organization of the genes for the following phosphagen kinases: Eisenia LK, Sabellastarte AK, and Arenicola mitochondrial TK (MiTK). Analysis of genomic database for the polychaete Capitella sp. yielded two putative LK genes [cytoplasmic LK and mitochondrial LK (MiLK)]. The intron/exon organization of these genes was compared with available data for cytoplasmic and mitochondrial CKs, and an annelid GK. Surprisingly, these annelid genes, irrespective of whether they are cytoplasmic (LK, AK, and GK) or mitochondrial (MiTK and MiLK), had the same 8-intron/9-exon organization and were strikingly similar to MiCK genes sharing seven of eight splice junctions. These results support the view that the MiCK gene is basal and ancestral to the phosphagen kinases unique to annelids.

To elucidate the relationship between dsx and mab-3 related transcription factor 1 (Dmrt-1) and differentiation, alteration in mRNA levels during differentiation of NEC8 human embryonic carcinoma cells was investigated. After stimulation with 50 nM phorbol 12-myristate 13-acetate (PMA), the cells differentiated into cells with mesenchymal characteristics and upregulated Dmrt-1 mRNA, possibly through the protein kinase C/mitogen-activated protein kinase/activated protein-1 signaling pathway. Conversely, knockdown of Dmrt-1 by small interfering RNA resulted in cell morphology that was different from that after PMA treatment. These results indicated that Dmrt-1 expression was apparently associated with the differentiation of NEC8, and this cell line may be a helpful in vitro tool to clarify the role of Dmrt-1 in the differentiation process., ((Cancer Sci 2006; 97: 277 -282).)

Two cDNAs similar to mammalian valosine-containing proteins (VCPs) were isolated from the common lumbricid earthworm Eisenia fetida (Savigny, 1826). The primary sequences, referred to as eVCP-1 and eVCP-2, display a similarity of 74%. Despite of the variable C-termini, both eVCPs have a conserved intron/exon organization spanning 14 kb, which is also conserved to their mammalian counterparts. Although this finding strongly suggests VCPs have a common ancestral origin, phylogenetic analysis predicts that eVCP-2 may be distinct. An investigation by reverse transcription-polymerase chain reaction (RT-PCR) revealed that, whilst evcp-1 was ubiquitously expressed during all developmental stages, evcp-2 was specifically expressed in the anterior segments of sexually mature earthworms. In situ hybridization clearly demonstrated that evcp-2 is expressed in the seminal vesicles, the location of spermatogenesis, and more precisely within the cytophores surrounded by secondary spermatocytes or spermatids. Taken together, this evidence leads to the notion that eVCP-2 is a likely component involved in the final modulation of spermatogenesis.

The epidermal growth factor receptor and its ligands initiate a major signaling pathway that regulates keratinocyte growth in an autocrine manner. It is well known that high doses of epidermal growth factor receptor ligands inhibit keratinocyte growth. Recently, signal transducers and activators of transcription 1-dependent p21Waf1/Cip1 induction were reported to be involved in high-dose epidermal growth factor-dependent cell growth arrest in the A431 squamous cell carcinoma cell line; however, transfection of dominant-negative signal transducers and activators of transcription 1 adenovirus vector did not block epidermal growth factor-induced growth inhibition in normal human keratinocytes. As transforming growth factor beta is a potent inhibitor of keratinocyte proliferation, we hypothesized that transforming growth factor beta contributes to epidermal growth factor-mediated keratinocyte growth inhibition. Epidermal growth factor concentrations of 10 ng per ml enhanced transforming growth factor beta1 mRNA expression from 3 to 6 h poststimulation. Enzyme-linked immunosorbent assay analysis detected 150 pg per ml of transforming growth factor beta1 in the culture medium of keratinocytes incubated with 10 and 100 ng per ml epidermal growth factor, whereas 0.1 and 1.0 ng per ml epidermal growth factor slightly enhance transforming growth factor beta1 production. Epidermal growth factor (100 ng per ml) upregulated luciferase activity of p3TP-lux, which contains three tandem transforming growth factor beta-Smad signaling responsive elements, 6-fold compared with unstimulated cells. The epidermal growth factor-dependent induction of p3TP-lux luciferase activity was disrupted by transfection of the dominant negative form of transforming growth factor beta type I receptor adenovirus vector (AxdnALK5), which suggests that epidermal growth factor-induced transforming growth factor beta acts in an autocrine manner in keratinocytes. Moreover, transfection of AxdnALK5 completely blocked the growth inhibition induced by 100 ng per ml of epidermal growth factor in normal keratinocytes. These data demonstrate that an autocrine transforming growth factor beta1-ALK5 pathway is a negative feedback mechanism for epidermal growth factor-induced normal human keratinocyte growth.

It is known that EGF induces the cell-cycle arrest in A431 cells that possess high numbers of EGF receptors and it was previously suggested that p21/WAF1 protein was a major effector molecule of the EGF-mediated cell-cycle arrest of A431 cells. Here, we further investigate this phenomenon using the decoy double-strand oligonucleotides for STAT-binding sequence (STAT decoy) and IkappaB, an inhibitor of the nuclear factor kappa B (NFkappaB). Addition of STAT decoy restored EGF-induced A431 cell-growth arrest. Interestingly, infection of adenovirus vectors to express IkappaB (AxIkappaBalphaDeltaN) as the inhibitor of NFkappaB also reversed the A431 cell-growth inhibition. The individual treatment of two inhibitors partially inhibited the WAF1 gene expression, whereas simultaneous treatment of two inhibitors exhibited more efficient inhibition. These observations suggest the activation of NFkappaB via IkappaB degradation and STAT1 via specific receptor kinase activity synergistically induce WAF1 gene expression in A431 cells. Thus, NFkappaB and STAT1 pathways mutually interact to play an important role in the EGF-induced intracellular reaction., (Copyright 2000 Wiley-Liss, Inc.)

The Fab fragment of monoclonal antibody B4G7 against human epidermal growth factor (EGF) receptor was conjugated with cationic poly-L-lysine and the resulting conjugate was further complexed with reporter genes or therapeutic genes. This Fab/DNA complex was designated as "Fab immunogene." The Fab immunogene transfer in vitro was mediated through the EGF receptors in two melanoma cell lines. The frequency of cells expressing beta-galactosidase (beta-Gal) reporter gene was approximately 1%. The induction of suicide effects after Fab immunogene transfer of herpes simplex virus thymidine kinase (TK) or Escherichia coli cytosine deaminase (CD) gene was quite remarkable, and the growth of melanoma cells was inhibited for over 7 days in the presence of ganciclovir (GCV) or 5-fluorocytosine (5-FC). Similarly, when melanoma cells treated in vitro with the Fab immunogene carrying TK or CD were transplanted into the back of nude mouse, subsequent systemic administration of GCV or 5-FC effectively suppressed the growth of tumors, indicating the occurrence of in vivo suicide effects.

The "Fab immunogene" is a novel gene transfer vehicle in which the Fab fragment of anti-human epidermal growth factor (EGF) receptor antibody B4G7 is conjugated with poly-L-lysine to form an affinity complex with DNA. It was developed to target delivery of therapeutic genes into EGF receptor-hyperproducing tumor cells. Various characteristic features of the immunogene have been documented (Chen et al., 1998). Here we add further evidence to prove that in vitro transfer of beta-galactosidase/Fab immunogene is exclusively to EGF receptor-positive cells and that the herpes simplex virus thymidine kinase (TK)/Fab immunogene induces substantial suicide effects on A431 tumor cells when treated together with ganciclovir. The in vivo specificity of the immunogene transfer was examined using A431 tumor-bearing nude mice. When these nude mice were injected intraperitoneally with the chloramphenicol acetyltransferase (CAT)/Fab immunogene, CAT DNA was detected in the tumors as well as in liver and kidney but not brain, whereas CAT mRNA and enzyme activity were detected only in the tumors. Local and intraperitoneal injection of the TK/Fab immunogene and subsequent administration of ganciclovir effectively suppressed the growth of A431 tumors transplanted on the backs of nude mice. These observations suggest a possible application of the Fab immunogene system in cancer gene therapy.

We previously developed the "immunogene" approach toward cancer gene therapy using epidermal growth factor receptor (EGFR)-mediated endocytosis. Here, we describe an improved immunogene system, in which the antigen-binding (Fab) fragments of the monoclonal antibody (Ab) B4G7 against the human EGFR were conjugated with poly-L-lysine to form a gene delivery vehicle (designated Fab "immunoporter"). Within 12 hours, the beta-galactosidase beta-gal) gene was transferred via the Fab immunoporter to virtually all of the nuclei of human squamous carcinoma A431 cells that overproduce the EGFR, and the beta-gal enzyme activity was detected within 24 hours and retained for more than 3 days. The beta-gal gene was not transferred into human and mouse cells that were deficient in EGFRs, but it was delivered if those mouse cells were transformed with human EGFR genes. Beta-gal gene transfer via the Fab immunoporter was inhibited by pretreatment with excess amounts of the Fab fragment. The transfer efficiency of the beta-gal gene to A431 cells via the Fab immunoporter was approximately 2%, which is as high as the lipofection method and 20- to 100-fold higher than the whole Ab immunoporter. The transfer of the herpes simplex virus thymidine kinase gene into A431 tumor cells as a form of the thymidine kinase/Fab immunogene was successful, and subsequent treatment with ganciclovir induced remarkable suicide effects which conferred 1000-fold higher drug sensitivity. Thus, the Fab immunogene was substantially improved with regard to the whole Ab immunogene and could be used as a potent gene transfer vehicle for the in vivo targeting of EGFR-hyperproducing tumor cells.

We are developing the "immunogene" system for the targeted delivery of therapeutic genes. The immunogene system utilizes the EGF receptor-mediated endocytosis. The Fab fragment of monoclonal antibody B4G7 against human EGF receptor was conjugated with polylysine to form an "Fab immunoporter", which forms an affinity complex with DNA. The transfection efficiency of Fab immunogene was approximately 10-fold higher than the Lipofectin. Gene transfer of HSV-tk gene into A431 tumor cells with Fab immunoporter was successful and the subsequent treatment with ganciclovir induced remarkable suicide effects conferring 1000-fold higher drug sensitivity. Thus, the immunogene system could be useful as a gene transfer vehicle targeting the EGF receptor-hyperproducing tumor cells.

A431 cells hyperproduce EGF receptors and possess inactive p53 proteins. It has been suggested that a cyclin-dependent kinase (CDK) inhibitor p21/WAF1 plays a crucial role in the EGF-induced cell-cycle arrest of A431 cells. Here, we investigated the role of WAF1 gene transcription in the EGF-induced cell-cycle arrest by transfecting the 18-mer antisense oligonucleotide which corresponds to the 5' region of WAF1 gene (AS/WAF1). When A431 cells were treated with EGF, a cascade of responses were observed, including immediate hyperphosphorylation of EGF receptor on tyrosine residues, accumulation of WAF1 mRNA and p21/WAF1 protein, dephosphorylation of RB protein which is a substrate of CDK-cyclin, and cell-cycle arrest. In the presence of AS/WAF1, EGF induced the tyrosine-phosphorylation of EGF receptor, but WAF1 mRNA was reduced to a half; accumulation of p21/WAF1 protein and its downstream responses were no longer observed; A431 cells grew continuously. Thus, the transfection of antisense efficiently prevented A431 cells from the EGF-induced arrest. These observations suggest that p21/WAF1 protein is a major effector molecule of the EGF-mediated cell-cycle arrest of A431 cells.

The members of EGF receptor subfamily are transmembrane glycoproteins with tyrosine kinase activity. Once EGF or EGF-related peptides binds to the receptors, they undergo autophosphorylation and binding to the SH2 protein, which in turn activates the intracellular signaling cascade. In the squamous carcinoma of head and neck, esophagus and lung, the EGF receptor gene amplification and EGF receptor hyperproduction are frequently observed. In the adenocarcinoma of pancreas, breast and stomach, hyperproduction of the EGF receptor subfamily is common, suggesting the involvement of these growth factor receptors in the tumorigenesis. The prognostic value of EGF receptor hyperproduction appears considerable when combined with other factors. EGF receptor subfamily members are useful targets for the immunotoxin therapy and immunogene therapy.

In this article we describe an improved method to produce a conjugate of anti-erythrocyte growth factor (EGF) receptor monoclonal antibody with polylysine via thio-ether bonds. The resulting antibody/polylysine conjugate was found to be a much more stable DNA (gene) carrier than the previous conjugate formed via disulfide bonds. We designated the conjugate as an "immunoporter" and the immunoporter/DNA (gene) complex as an "immunogene." The fluorescent microscopic observation showed that the immunoporter as well as immunogene bound specifically to the EGF receptors on the cell surface, and the loaded reporter gene, such as beta-galactosidase (beta-GAL), was detected in the cell nucleus at 2 hours after transfection. The enzyme activity from the beta-GAL gene was detected at 12 hours and increased for 3 to 5 days. Similar kinetics were obtained for another reporter gene, chloramphenicol acetyltransferase. Furthermore, the immunoporter delivered the herpes simplex virus thymidine kinase gene and induced substantial suicide effects on tumor cells when gancyclovir or acyclovir was added. Thus, the immunogene approach was successful in delivering therapeutic genes to EGF receptor overexpressing tumor cells. Further technical refinement may prove useful as a supplementary treatment of patients with squamous cell carcinomas.

The target molecule of pemphigus autoantibodies is a transmembrane desmosomal component, desmoglein 3 (Dsg3) in pemphigus vulgaris (PV) and Dsg1 in pemphigus foliaceus (PF). In this study, we examined the effects of calcium and glycosylation on the anti-genicity of the pemphigus antigens and on the generation of conformational epitopes. We used recombinant baculovirus proteins, PVIg and PFIg, which are considered to reflect accurately the native conformation of the extracellular domain of their respective proteins Dsg3 and Dsg1. These baculoproteins could immunoadsorb heterogeneous autoantibodies from the corresponding sera of PV and PF patients, completely blocking indirect immunofluorescence staining of normal human skin. Chelating calcium from the solution containing the baculoproteins using ethylenediaminetetraacetic acid (EDTA) or ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) abolished immunoadsorption by both PVIg and PFIg; however, immunoadsorption by the baculoproteins was restored after dialysis against 1 mM calcium. Nonglycosylated forms of both baculoproteins produced in the presence of tunicamycin retained their immunoadsorptive ability. Furthermore, immunoadsorption by the baculo-proteins was prevented irreversibly by treatment with low pH, high pH, and boiling, but not with the non-ionic detergent Nonidet P-40. These findings indicate that formation of the conformational epitopes on the pemphigus antigens is dependent on calcium but independent of glycosylation, and provide direct evidence that calcium plays an important role in determining the antigenic properties of the pemphigus antigens.

We investigated the antigen molecules for six clinically typical cases of paraneoplastic pemphigus (PNP) using immunofluorescence, immunoprecipitation, and immunoblotting. All the PNP sera showed a clear reactivity with transitional epithelia of rat urinary bladder and immunoprecipitated the 250-kD, 230-kD, 210-kD, 190-kD, and 170-kD proteins in various combinations, confirming the diagnosis of PNP. Immunoblot analysis demonstrated slightly different reactivity from that of immunoprecipitation. With immunoblotting of normal human epidermal extract, bovine desmosome preparation, and extract of cultured squamous cell carcinoma cells, all the PNP sera reacted with a characteristic doublet of the 210-kD and 190-kD proteins. However, immunoblotting detected the 250-kD desmoplakin I and the 230-kD bullous pemphigoid antigen less frequently and did not detect the 170-kD protein. Further immunoblot studies indicated that the 210-kD protein is different from desmoplakin II and that the 190-kD protein is most frequently detected by PNP sera. Two of the six PNP sera specifically reacted with the extracellular domain of recombinant pemphigus vulgaris antigen protein, indicating that pemphigus vulgaris antigen may be involved in PNP. In future studies to unravel the complex mechanisms of the PNP antigens, the immunoblot technique may be a useful tool.

Desmoyokin was identified as a desmosomal plaque protein. We previously demonstrated that desmoyokin is identical to a protein encoded by a human gene, AHNAK, whose expression is suppressed in neuroblastoma cells. Although this protein is distributed in the cytoplasm and the nucleus in various cells, it is associated closely with the plasma membrane in keratinocytes. In keratinocytes, desmoplakin translocates from the cytoplasm to the plasma membrane following both high calcium switch and protein kinase C (PKC) activation by 12-O-tetradecanoylphorbol-13-acetate (TPA). In the low calcium medium, the desmoyokin/AHNAK protein resides diffusely in the cytoplasm and the nucleus. However, 2 h after shift to the high calcium medium, the desmoyokin/AHNAK protein localized to the cell boundary in all cells in a pattern similar to that of desmoplakin. Selective PKC inhibitors completely inhibited the calcium-induced translocation of the desmoyokin/AHNAK protein, but the inhibition of desmoplakin translocation by these inhibitors was only partial. TPA also induced translocation of both the desmoyokin/AHNAK protein and desmoplakin, which was completely inhibited by PKC inhibitors. The calcium-induced phosphorylation of the desmoyokin/AHNAK protein was confirmed by immunoprecipitation using [32P]orthophosphate-labeled keratinocytes. Furthermore, the study of extractability with non-ionic detergent indicated that desmoplakin, but not the desmoyokin/AHNAK protein, is associated with the cytoskeleton. These results suggested an involvement of PKC in the translocation of the desmoyokin/AHNAK protein in keratinocytes. It was, however, also suggested that different mechanisms are likely involved in the translocation of the desmoyokin/AHNAK protein and desmoplakin.

We found that hydrogen peroxide (H2O2) enhances EGF receptor tyrosine phosphorylation in intact cells as well as solubilized membrane of an EGF receptor hyperproducing cell line NA. An antioxidant MnCl2 effectively inhibited this enhancement. Interestingly, overall phosphorylation of the EGF receptor enhanced by H2O2 was half that of the EGF-enhanced phosphorylation when the receptor immunoprecipitated from [32P]orthophosphate-labeled cells was examined. Tryptic phospho-peptide mapping of these receptors revealed that EGF enhanced the phosphorylation on five specific residues including serine 671, 1,046 and 1,047, threonine 669 and tyrosine 1,173, whereas H2O2 enhanced the phosphorylation remarkably on tyrosine 1,173 and three other residues and only moderately on serine 1,046 and 1,047 and threonine 669. Thus, H2O2 preferentially enhances the tyrosine phosphorylation of EGF receptor through oxidant stress.