Internucleosomal DNA fragmentation during phorbol ester-induced monocytic differentiation and G0/G1 arrest.

The treatment of human myeloid leukemia cell lines with phorbol esters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), is associated with loss of proliferative capacity and induction of monocytic differentiation. The present results demonstrate that treatment of asynchronous human U-937 leukemia cells with 10 nM TPA is also associated with oligonucleosomal DNA cleavage. This pattern of DNA fragmentation, which is observed in programmed cell death, was detectable in populations of TPA-treated cells that had entered a nonproliferative G0/G1 phase. Similar findings were obtained after TPA treatment of a synchronous population of G1 cells. These cells progressed through S and G2/M phases before undergoing internucleosomal DNA cleavage during G0/G1 arrest. These G0/G1 cells displayed characteristics of monocytic differentiation, including down-regulation of c-myc expression and induction of c-fms transcripts. DNA fragmentation was also studied in cells treated with 5 nM TPA for 48 h and then monitored in drug-free long-term culture. Endonucleolytic cleavage was similarly observed in the differentiated G0/G1 population. However, longer periods of culture were associated with a decrease in DNA fragmentation to undetectable levels. This effect was followed by retrodifferentiation and reentry of cells into cycle. Taken together, these findings demonstrate that internucleosomal DNA fragmentation occurs during induction of monocytic differentiation, and that both of these events are detectable in G0/G1 cells.

Ionizing radiation induces expression and binding activity of the nuclear factor kappa B.

Recent studies have demonstrated that treatment of mammalian cells with ionizing radiation is associated with activation of gene expression. Although the signal transduction pathways stimulated by ionizing radiation remain unclear, our previous findings indicate that radiation induces specific genes at the transcriptional level. The present work has examined the effects of ionizing radiation on the transcription factor NF-kappa B. The results demonstrate that ionizing radiation activates DNA binding of nuclear factor (NF)kappa B. This effect was detectable at 2 grays (Gy) and reached a maximum at 5-20 Gy. At a dose of 20 Gy, the increase in NF-kappa B binding activity was maximal at 2-4 h and then declined to pretreatment levels. The results also demonstrate that ionizing radiation transiently increases NF-kappa B mRNA levels. However, the finding that induction of NF-kappa B binding to DNA occurs in the presence of cycloheximide indicates that ionizing radiation activates preexisting NF-kappa B protein. NF-kappa B exists as a cytoplasmic protein before activation. Thus, our results suggest that ionizing radiation induces transduction pathways which include cytoplasmic signaling events.

Laparoscopy-assisted hepatic lobectomy using hilar Glissonean pedicle transection.

Although many reports have described laparoscopic minor liver resections, major hepatic resection, including right or left lobectomy, has not been widely developed because of technical difficulties. This article describes a new technique for performing laparoscopy-assisted right or left hepatic lobectomy using hilar Glissonean pedicle transection. Laparoscopic mobilization of the right or left hepatic lobe is performed, including dissection of the round, faliciform, triangular, and coronary ligaments. The right or left Glissonean pedicle is encircled and divided laparoscopically. A parenchymal dissection is then performed though the upper median or right subcostal incision, through which the resected liver is removed. We successfully performed this procedure in 6 patients without blood transfusion or serious complications. Laparoscopy-assisted hepatic lobectomy using hilar Glissonean pedicle transection can be feasible and safe in highly selected patients.

Induction of internucleosomal DNA fragmentation in human myeloid leukemia cells by 1-beta-D-arabinofuranosylcytosine.

The present results demonstrate that treatment of human U-937 myeloid leukemia cells with 1-beta-D-arabinofuranosylcytosine (ara-C) is associated with DNA fragmentation at multiples of approximately 200 base pairs. The extent of ara-C-induced DNA fragmentation was dependent on drug concentration and time of exposure. This pattern of internucleosomal DNA cleavage has been observed during programmed cell death and was associated in the present studies with loss of clonogenic survival. The results also demonstrate that the c-jun protooncogene is induced by ara-C during periods of DNA cleavage. These findings suggest that ara-C activates a program involving both oligonucleosomal DNA fragmentation and changes in early response gene expression.

cis-Diamminedichloroplatinum(II) induces c-jun expression in human myeloid leukemia cells: potential involvement of a protein kinase C-dependent signaling pathway.

cis-Diamminedichloroplatinum(II) (CDDP) is a chemotherapeutic agent known to inhibit DNA, RNA, and protein synthesis. The cytotoxicity of this drug is thought to result from the formation of DNA intrastrand cross-links. The present work demonstrates that treatment of human myeloid leukemia cells (HL-60, U-937, and KG-1) with CDDP is associated with increased expression of the c-jun gene and that this effect is related to activation by a transcriptional mechanism. The results also demonstrate that treatment with CDDP is associated with increases in protein kinase C (PKC) activity. Furthermore, the finding that pretreatment with H7, an inhibitor of PKC, abrogates the effect of CDDP on c-jun expression suggested the involvement of PKC in this process. Down-regulation of PKC by prolonged pretreatment with 12-O-tetradecanoylphorbol-13-acetate was also associated with inhibition of CDDP-induced c-jun expression. The results further demonstrate that there is a temporal relationship between the CDDP-induced increase in c-jun expression and the occurrence of internucleosomal DNA cleavage characteristic of programmed cell death. These findings suggest that c-jun may be involved in the cellular response to DNA-damaging agents, such as CDDP, and that this effect may be mediated by a PKC-dependent pathway.

Differentiation and retrodifferentiation of human myeloid leukemia cells is associated with reversible induction of cell cycle-regulatory genes.

Treatment of human myeloid leukemia cells (HL-60, U-937, THP-1) with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with differentiation along the monocytic lineage. This induction by TPA is characterized in part by growth arrest and the appearance of differentiated monocytic phenotype. The present studies demonstrate that myeloid leukemia cells exit the cell cycle to G0-G1 between 24 and 36 h following TPA treatment. This G0-G1 arrest was accompanied by down-regulation of the cell cycle-regulatory genes cdc2, cyclin A, cyclin B, and cdc25. Similar findings were obtained for histones H1 and H4. Cell cycle progression of synchronized U-937 cells revealed low to undetectable mRNA levels for these genes in G1 and maximal transcription in G2-M phase. Results obtained from mRNA half-life studies demonstrate that the stability of cdc2, cyclin A, cyclin B, and cdc25 transcripts is similar in control and TPA-treated U-937 cells. Nuclear run-on assays demonstrated down-regulation of histone gene transcription, while there was no signal detectable for the cell cycle-regulatory genes. The present findings also demonstrate that long term culture of TPA-differentiated U-937 cells is associated with a decrease in G0-G1-arrested cells and an increase of cells in S and G2-M after 25 days. This reentry into the cell cycle was accompanied by loss of adherence, down-regulation of markers for the monocytic phenotype, and induction of the cell cycle-regulatory genes. This process of retrodifferentiation was completed after 36 days when patterns of cell cycle-regulatory and histone gene expression were identical to that in untreated U-937 cells.

Camptothecin and its derivatives induce expression of the c-jun protooncogene in human myeloid leukemia cells.

We have recently demonstrated that certain camptothecin derivatives are effective agents in the treatment of human tumor xenografts in nude mice. While camptothecin and its derivatives are recognized as inhibitors of topoisomerase I, little is known about the effects of these agents on specific gene expression, particularly genes involved in growth control. The c-jun early response gene codes for a leucine zipper transcription factor. The present studies demonstrate that 20(S)-camptothecin, 9-amino-20(S)-camptothecin, and 9-nitro-20(S)-camptothecin inhibit the growth of human U-937 myeloid leukemia cells and induce expression of the c-jun gene. c-jun transcripts were increased at 3 h and reached a maximum at 6 h of drug exposure. We also demonstrate that the induction of c-jun gene expression by these agents occurs at the transcriptional level. H7, a nonselective inhibitor of protein kinase C, completely blocked c-jun expression in 20(S)-camptothecin-treated cells, while another protein kinase inhibitor, HA1004, had no detectable effect. Similar findings were obtained for other leucine zipper encoding genes, including jun-B. These results suggest that 20(S)-camptothecin, 9-amino-20(S)-camptothecin, and 9-nitro-20(S)-camptothecin activate a cellular response involving the induction of early response genes. Finally, we demonstrate that induction of c-jun expression occurs in association with internucleosomal DNA fragmentation, a characteristic of programmed cell death.

Inhibition of EGR-1 and NF-kappa B gene expression by dexamethasone during phorbol ester-induced human monocytic differentiation.

The treatment of human myeloid leukemia cells (HL-60, U-937, THP-1) with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with growth arrest and appearance of a differentiated monocytic phenotype. While previous studies have reported that the glucocorticoid dexamethasone blocks phenotypic characteristics of monocytic differentiation, we demonstrated in the present work that dexamethasone delays the effects of TPA on the loss of U-937 cell proliferation. We also demonstrated that this glucocorticoid inhibits TPA-induced increases in expression of the EGR-1 early response gene. The results of nuclear run-on assays and half-life experiments indicated that this effect of dexamethasone is regulated at the post-transcriptional level. Similar studies were performed for the NF-kappa B gene. While TPA treatment was associated with transient increases in NF-kappa B mRNA levels, this induction was blocked by dexamethasone. In contrast, dexamethasone had no significant effect on the activation of pre-existing NF-kappa B protein as determined in DNA-binding assays. Taken together, these findings suggest that the activated glucocorticoid receptor inhibits signaling pathways which include expression of the EGR-1 and NF-kappa B genes and that such effects may contribute to a block in TPA-induced monocytic differentiation.

Effects of cilostazol lotion on blood flow in rabbit skin.

Cilostazol (Cls) is a inhibitor of phosphodiesterase and increases cyclic AMP (cAMP) in platelets and also raises the vascular smooth muscle cell cAMP level causing vasodilation. Therefore, it was expected to increase local blood flow in the skin. Topical application of Cls may improve local blood flow without systemic effects in clinical situations. In this paper the effect of Cls lotion on skin blood flow was assessed in animal experiments. Application of this lotion allowed skin blood flow to remain at increased levels for about 60-90 min. Tissue assay of the Cls content revealed that Cls is absorbed percutaneously and retained, even in the inner tissue layer, for at least 180 min.

Effects of prostaglandin E1 on cultured dermal fibroblasts from normal and hypertrophic scarred skin.

To investigate the role of prostaglandin (PG) E1 in preventing scar formation as well as that of the related cytokines, we culture fibroblasts from hypertrophic scar tissue (SDF) and normal dermis (NDF) collected from patients with scar contracture. We have compared the type I collagen synthesis, type I collagenase activity, and the production of interleukin (IL)-6, IL-8 and transforming growth factor (TGF)-beta(1) in two types of cultured fibroblasts before and after addition of PGE1. Our results demonstrated that levels of type I collagen and TGF-beta(1) production were higher and that type I collagenase activity and IL-8 production were significantly lower in the culture supernatants of SDF. There was no significance difference in IL-6 production between SDF and NDF culture supernatants. On the other hand, PGE1 significantly increased type I collagenase activity and IL-8 production in the SDF culture supernatants and it increased IL-6 and TGF-beta(1) production in both types of fibroblasts. However, there was no effect on synthesis of type I collagen in either group. To further investigate the role of TGF-beta(1) in NDF and SDF, exogenous recombinant human (rh) TGF-beta(1) was added. In NDF group, rhTGF-beta(1) induced a decrease in the type I collagenase/type I collagen ratio, while rhTGF-beta(1) had no effect on the same ratio in the SDF group. These results suggest that PGE1 may have a role in the prevention of hypertrophic scar by increasing the activity of type I collagenase.

Studies on cytokines related to wound healing in donor site wound fluid.

This study was performed to evaluate cytokines in donor-site wound fluids and to determine their effect on wound healing. A film dressing was applied to the donor-site wound of 24 patients immediately after a split-thickness skin graft was taken. On the 5th day after treatment, 2-3 ml of the fluid retained under the film dressing was collected by means of puncture with a syringe. Growth factors and cytokines considered to accelerate wound healing were present in relatively large amounts in the exudate. Very low concentrations of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were detected by a commercially-available enzyme-linked immunosorbant assay (ELISA) kit. However, the presence of both growth factors in wound fluid could not be confirmed because of the possible cross-reactivity of the antibodies to other EGF and FGF family growth factors. In contrast, platelet derived growth factor (PDGF), interleukin-6 (IL-6), transforming growth factor-alpha (TGF-alpha) and TGF-beta were present in relatively large amounts. The finding that certain cytokines coexist in a balanced state under the film dressing suggests that epithelization can proceed, since an adequate balance would insure proper regulation by the cytokine network. Our present study increases the likelihood that film or hydrocolloid dressings will be used more frequently in the future for treatment of burn wounds, ulcers or donor-site wounds since these dressings were shown to be more capable than ointments of retaining cytokines, particularly intrinsic growth factors secreted at the wound site.

Semen specific gamma-glutamyltransferase carries ABH antigens: a sandwich ELISA for simultaneous semen detection and its ABO blood typing.

Semen type of gamma-glutamyltransferase (gamma-GTP) is different from the membrane bound type of the enzyme in both biochemical and immunological properties, and consists of two subunits (150 and 95 kDa). We found that anti-ABH antibodies recognize a 150-kDa subunit of seminal gamma-GTP by Western blot and immunoprecipitation analyses. Using SG2, one of anti-semen specific gamma-GTP monoclonal antibodies which we had produced, and anti-ABH antibodies, we established a sandwich ELISA for identifying human seminal gamma-GTP and its ABO type simultaneously. This sandwich ELISA allows ABO typing of highly diluted semen. The dilutions for ABO typing were 10(5) times for A or O, and 10(4) times for B. Furthermore, ABO typing of semen was successfully performed by this ELISA, even in the mixed presence of vaginal fluid, saliva and blood. Thus, seminal gamma-GTP carries ABH antigens and the sandwich ELISA with SG2 and anti-ABH antibodies enables ABO typing of semen. The sandwich ELISA is extremely useful for ABO typing originated from semen in the mixture of biological fluids.

A sandwich enzyme-linked immunosorbent assay for human seminal gamma-glutamyl transpeptidase.

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detecting human seminal gamma-glutamyl transpeptidase (gamma-GTP) using a combination of anti-seminal gamma-GTP monoclonal antibodies. These monoclonal antibodies did not react with human ovary or uterus in immunohistochemical study. Optimal assay condition, resulting in a sensitive assay with a low background, is presented. The detection limit of this assay was estimated to be 1 ng/ml of seminal gamma-GTP corresponding to about 100,000 times dilution of seminal sample. This ELISA was specific for seminal gamma-GTP, without cross-reactivity to renal or hepatic gamma-GTP, normal blood serum, non-coital vaginal fluid or saliva. The recovery of seminal gamma-GTP added to various biological fluids were also examined.

Clinical effectiveness of an ointment containing prostaglandin E1 for the treatment of burn wounds.

This report discusses the effectiveness of conservative treatment of burns using applications of prostaglandin E1 (PGE1) containing ointment (PGE1 ointment) to the wound site. Fourteen patients with superficial dermal burns (SDB), deep dermal burns (DDB) and full-thickness dermal burns (DB), who were treated with repeated applications of this ointment, showed rapid epithelialization of the affected tissue. Hypertrophic scarring after epithelialization was less than that expected after other therapies. The degree of scarring was graded as 'none', 'mild', 'moderate' or 'severe'. The results of this therapy revealed no scarring in two sites (8.7 per cent), mild scarring in 16 sites (69.6 per cent), moderate scarring in one site (4.3 per cent) and severe scarring in four sites (17.4 per cent) out of 23 sites distributed among the patients. The application of PGE1 ointment in combination with skin grafting surgery was found to improve functional and aesthetic results in the patients with DDB and DB, by minimizing the area of the donor site; it was especially useful for children with extensive burns because of the shortage of available tissue as donor site material for skin grafting.

A study of cytokines in burn blister fluid related to wound healing.

This report indicates that retention fluid from blisters of partial skin thickness burns, which contains relatively large amounts of cytokines and growth factors, stimulates the wound healing process. Although epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) levels were low, relatively large amounts of cytokines including platelet derived growth factor (PDGF), interleukin (IL-6) and transforming growth factor (TGF) alpha were present and these exercised stimulatory effects on wound healing. TGF beta, which plays an important role in collagen metabolism and in scar formation, was also detected. Contrary to our expectations, IL-1 alpha and beta, both of which initiate inflammation, were detected at relatively low levels whereas IL-8 levels were rather high. Various cytokines were shown to coexist in a balanced state in the retention fluids, suggesting that epithelialization might be regulated via a cytokine network operating on the wound surface. The growth of keratinocytes in culture significantly increased with the addition of 1 per cent or more of blister fluid to the medium.

Studies of tissue cultured sliced dermis as a skin substitute.

A dermis slicer designed by the authors enabled us to prepare about 10 sheets of sliced dermal grafts (SDG), 300 micron(s) thick, from the dermis harvested from the back or buttocks of adult patients during operations. Such a sliced dermal sheet was stretched with one surface stuck on the base of a culture dish. It was then incubated in Dulbecco's essential medium for tissue culture, to which epidermal growth factor had been added. By the first week only its upper side was epithelialized from epithelial components in sliced dermis. The formation of basement membrane with anchoring fibrils was confirmed by electron microscopy. The appearance of type IV collagen and laminin was observed between epithelialized basal cells and the dermal layer. Thus, it is thought that the SDG is useful not only for immediate grafting, when epithelialization follows, but also as a substitute for free split thickness skin grafts following tissue culture.

Effects of a platelet activating factor antagonist on oedema formation following burns.

Deep dermal burns covering 30 per cent of the total body surface area were prepared by immersing the backs of rabbits in hot water at 80 degrees C for 20 s, to determine whether platelet activating factor (PAF) was involved in the onset of oedema following burns and to evaluate the effect of TCV-309, a potent PAF antagonist. The PAF antagonist, which was infused soon after the burn, blocked oedema formation in the wound and significantly (P < 0.001) inhibited PAF increase (P < 0.05) in the damaged tissue in a dose-dependent manner. This was confirmed by immunohistochemistry. In contrast, the superoxide dismutase content in the group treated with a high dose of TCV-309 was significantly (P < 0.01) higher than that of the control group. These findings suggest that administration of large doses of a PAF antagonist immediately after injury prevents oedema of burn wounds and the subsequent onset of burn shock by suppressing PAF and superoxide radical formation.

Use of the oblique island flap in excision of small facial tumors.

Reconstruction with the oblique sigmoid island flap, which we contrived as a subcutaneous pedicle flap, is performed as follows: the small triangular skin flaps, about 1 to 2 mm in length on each side, are excised from both sides of a round defect following tumor excision in a direction corresponding to the wrinkle line, and a subcutaneous island flap which has a width equal to the defect and has shifted about half the distance of the defective area in parallel with the wrinkle line is advanced and sutured. Our technique is thought to be highly effective from a cosmetic point of view because reconstruction of the defects following excision of small tumors that occur in sites with delicate eminence, such as the area between the inner canthal region and the cheek, the site along the nasolabial fold, and around the alae, often tends to fail to achieve cosmetically good results when conventional local skin flaps are used.

Reconstruction of defects of the entire vermilion with a buccal musculomucosal flap following resection of malignant tumors of the lower lip.

We reconstructed a defect of nearly the entire lower vermilion using a buccal musculomucosal flap following resection of a malignant tumor of the lower lip and obtained satisfactory results. The buccal musculomucosal flap was semi-spindle shaped and pedicled at the angle of the mouth. A flap measuring as much as 1.5 cm in width and 5 cm in length could be raised while ensuring that fibers of the buccinator muscle extended over its entire length. Using this technique, it was possible to reconstruct a wide defect following tumor resection and removal of almost the entire lower vermilion by means of only a transposition of a unilateral buccal musculomucosal flap after about one-quarter of the lower lip had been excised and sutured primarily. Reconstruction with this technique is a two-stage operation, and a secondary minor touch-up operation is performed on the angle of the mouth at the same time as repair of the dog-ear of the pedicle. Advantages of this technique are that food can be taken orally soon after the operation, hemodynamics in the flap are maintained stably because the flap contains fibers of the buccinator muscle, and the vermilion is given a natural eminence. In addition, postoperative drooling is minimized, and sensation returns to the vermilion within the early postoperative period. Based on these advantages, we think our technique should be the first choice for carrying out reconstruction of defects that are located mainly in the lower lip vermilion because this is a safe and reliable method with which we performed 12 cases of vermilion reconstruction without flap necrosis and with satisfactory aesthetic and functional results.

A method of treatment of constricted ears with a conchal cartilage graft to the posterior auricular plane.

In order to make natural auricular features in a simple and certain way and also to prevent the occurrence of a postoperative lop-eared deformity, we contrived a new method to treat constricted ears. Our method consists of correction of the anthelical deformity according to a modified Converse method and a cartilage graft harvested from the posterior region of the concha to fix as a support on the posterior auricular plane which enables the auricle to maintain a normal shape. This method resulted in very satisfactory treatment of prominent ears or ears with mild helical constriction, categorized as group I, IIa, or IIb on Tanzer's classification, and the patients did not have marked postoperative problems. Thus, in this respect, our technique is considered to be a very effective and safe method.