Features of short-term myocardial hibernation

Infrared (IR) spectroscopy was used to compare the drug resistance mechanism of cells from chronic lymphocytic leukemia (CLL) patients with that of WSU-CLL cells. Bryostatin 1 (Bryo 1), a macrocyclic lactone and protein kinase C activator, was used to render WSU-CLL cells more susceptible to 2-chlorodeoxyadenosine (2-CdA). The IR spectroscopic analysis revealed some changes in protein and DNA content in Bryo 1-treated WSU-CLL cells, however, the most significant alterations were observed in the membrane lipids, which resemble those found between 2-CdA-sensitive and 2-CdA-resistant cells from CLL patients. In addition, Bryo 1 treatment induced WSU-CLL cells to become CD11c, CD25 and tartrate-resistant acid phosphatase-positive, specific markers for hairy cell leukemia, a disease exquisitely sensitive to 2-CdA. Our results suggest that 2-CdA-sensitive CLL cells have cellular characteristics resembling the hairy cell stage. The similarity between the membrane lipids in 2-CdA-sensitive CLL cells and the Bryo 1-treated WSU-CLL cell line supports the suggestion that membrane lipid alteration might be an important step in the drug resistance mechanism of CLL cells.     

2',2'-Difluorodeoxycytidine (gemcitabine) induces apoptosis in myeloma cell lines resistant to steroids and 2-chlorodeoxyadenosine (2-CdA)

The paucity of effective cytotoxic agents for the treatment of steroid resistant multiple myeloma explains the ongoing search for alternative substances for chemotherapy of this disease. In the present study, the purine antagonist 2-chlorodeoxyadenosine (2-CdA, cladribine) and the pyrimidine antagonist 2',2'-difluorodeoxycytidine (gemcitabine) were tested on four myeloma cell lines (i.e., U 266, OPM 2, RPMI 8226, IM 9), one plasma cell leukemia cell line (HS Sultan) and a myeloid control cell line (HL 60), all of which are resistant to 10-6 M dexamethasone. Gemcitabine has been found to be promising in the chemotherapy of other tumors with low proliferative activity, but its effectiveness against myeloma cells has not been analyzed so far. In our tests, gemcitabine induced a significant degree of apoptosis in all cell lines investigated. After incubation for 48 h with 10 microM gemcitabine, the median numbers of apoptotic cells were in the range of 45% in the OPM 2 and 79% in the U 266 cell line. All of the investigated cell lines were responsive to concentrations of 10 microM gemcitabine even after an exposure of only 30 min, three of them (U 266, HS Sultan, IM 9) also responded to a concentration of 10 nM. Higher concentrations and longer exposure times were necessary to suppress the growth of normal hematopoietic bone marrow progenitor cells. In contrast to gemcitabine, standard concentrations of 2-CdA (i.e., 30 and 300 nM) failed to induce a significant degree of apoptosis in the cell lines investigated but inhibited the growth of myeloid progenitor cells. The results suggest that gemcitabine induces apoptosis in myeloma and plasma cell leukemia lines resistant to steroids and 2-CdA. The fact that tumor cell apoptosis was achieved at concentrations clinically achievable and tolerable, which at the same time do not inhibit the growth of normal CFU-GM progenitor cells, favors the initiation of phase I trials with this drug for the treatment of multiple myeloma.     

2-Chlorodeoxyadenosine (2-CdA) in the treatment of patients with relapsed chronic lymphocytic leukemia

This study attempts to characterize the response of patients with chronic lymphocytic leukemia (CLL) to the purine analog 2-chlorodeoxyadenosine (2-CdA). We have treated 10 patients with 2-CdA, at a dose 0.05-0.1 mg/kg/daily, for 7 days as a 2-hour infusion. Mean age was 54.6 years (range, 34-68 years). Mean time from diagnosis to treatment with 2-CdA was 37.0 months (range, 8-84 months). All the studied patients had received preliminary therapy consisting of other than 2-CdA chemotherapeutic regimens. Eight out of 10 patients had Rai stage III-IV disease. Four patients had Coombs positive hemolytic anemia before 2-CdA treatment. Seven patients responded to 2-CdA. Two complete remission (CR) and 5 partial remission (PR) were achieved. All patients but one with Coombs positive autoimmune positive hemolytic anemia achieve complete resolution of hemolysis. Severe neutropenia was frequent, and serious infections were noted in 20%, 43% and 50% of cases during the first, second and third course of 2-CdA, respectively. We conclude that 2-CdA is an effective agent in relapsed CLL patients, particularly in cases complicated by autoimmune hemolytic anemia.     

Estrogenic effects of genistein on the growth of estrogen receptor-positive human breast cancer (MCF-7) cells in vitro and in vivo

Genistein, found in soy products, is a phytochemical with several biological activities. In the current study, our research focused on the estrogenic and proliferation-inducing activity of genistein. We have demonstrated that genistein enhanced the proliferation of estrogen-dependent human breast cancer (MCF-7) cells in vitro at concentrations as low as 10 nM, with a concentration of 100 nM achieving proliferative effects similar to those of 1 nM estradiol. Expression of the estrogen-responsive gene pS2 was also induced in MCF-7 cells in response to treatment with a concentration of genistein as low as 1 microM. At higher concentrations (above 20 microM), genistein inhibits MCF-7 cell growth. In vivo, we have shown that dietary treatment with genistein (750 ppm) for 5 days enhanced mammary gland growth in 28-day-old ovariectomized athymic mice, indicating that genistein acts as an estrogen in normal mammary tissue. To evaluate whether the estrogenic effects observed in vitro with MCF-7 cells could be reproduced in vivo, MCF-7 cells were implanted s.c. in ovariectomized athymic mice, and the growth of the estrogen-dependent tumors was measured weekly. Negative control animals received the American Institute of Nutrition (AIN)-93G diet, the positive control group received a new s.c. estradiol (2 mg) pellet plus the AIN-93G diet, and the third group received genistein at 750 ppm in the AIN-93G diet. Tumors were larger in the genistein (750 ppm)-treated group than they were in the negative control group, demonstrating that dietary genistein was able to enhance the growth of MCF-7 cell tumors in vivo. Increased uterine weights were also observed in the genistein-treated groups. In summary, genistein can act as an estrogen agonist in vivo and in vitro, resulting in the proliferation of cultured human breast cancer cells (MCF-7) and the induction of pS2 gene expression. Here we present new information that dietary genistein stimulates mammary gland growth and enhances the growth of MCF-7 cell tumors in ovariectomized athymic mice.     

Bryostatin 1 induces ubiquitin COOH-terminal hydrolase in acute lymphoblastic leukemia cells

It has been previously reported that Bryostatin 1 (Bryo1) induces differentiation of the human acute lymphoblastic leukemia (ALL) cell line, Reh, to a monocytoid B-cell stage. In this study we demonstrate that a novel protein, ubiquitin COOH-terminal hydrolase (UCH-L1), is associated with this differentiation. Reh cells were treated with 200 nmol/l of Bryo1 for 72 h and analyzed for changes in morphology, surface immunophenotype, acid phosphatase and terminal deoxynucleotidyl transferase. Protein patterns of the parent and differentiated cells, by two-dimensional polyacrylamide gel electrophoresis (2D PAGE), were studied. Bryo1-treated cells expressed morphologic, phenotypic and enzymatic features of the monocytoid B-cell stage. The UCH-L1 enzyme (MW-pl 34-5.3) was detected by 2 D PAGE in the differentiated, but not in parent cells. The presence of UCH-L1 in the Bryo1-treated cells was further confirmed by immunoblotting of 2 D PAGE using UCH-L1 polyclonal antibody. Ubiquitin expression was studied in parent and Bryo1-treated cells and was compared with 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated cells. Both agents, TPA and Bryo1, increased the level of ubiquitin expression as detected by flow cytometry. Sodium borohydride, an inhibitor of UCH-L1, inhibited the Bryo1-induced differentiating effect on Reh cells. To date, the mechanism by which Bryo1, exerts its B-cell differentiating effect is not fully understood. This study shows that UCH-L1 expression may play a major role in Bryo1-induced differentiation in pre-B-ALL.     

Increased interleukin-8 release by beta-adrenoceptor activation in human transformed bronchial epithelial cells

1. The effect of beta-adrenoceptor activation on release of the neutrophil chemoattractant, interleukin-8 (IL-8), was examined in human transformed bronchial epithelial cells (16HBE cells). 2. The combined beta 1- and beta 2-adrenoceptor agonist, isoprenaline, time- (100 nM, 2-18 h) and concentration- (1-30 nM) dependently increased IL-8 protein content in the cell culture supernatant as measured by an enzyme immunosorbent assay standardized for DNA by fluoro-colorimetry. 3. Isoprenaline (1-100 nM, 15 min) increased cyclic AMP concentration-dependently. 4. The effect of isoprenaline (100 nM) was inhibited by the beta-adrenoceptor blocker propranolol (10 microM). The maximum magnitude of IL-8 increase caused by beta-adrenoceptor activation was 40% of that caused by the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha 100 ng ml-1). 5. The selective beta 2-adrenoceptor agonist salbutamol (1 microM), increased IL-8 protein similarly to isoprenaline and the cyclic AMP analogue, dibutyryl cyclic AMP (1 mM) produced a corresponding effect. 6. Pretreatment with isoprenaline (100 nM) followed by TNF-alpha (20 ng ml-1) increased IL-8 additively. 7. In conclusion, beta-adrenoceptor stimulation increased the release of the neutrophil chemoattractant, IL-8 in 16HBE cells, via an increase in intracellular cyclic AMP. beta-adrenoceptor stimulation adds to the IL-8 increase caused by the pro-inflammatory cytokine TNF-alpha. If this mechanism exists in vivo, beta-adrenoceptor activation may increase neutrophil chemotaxis into the airways.     

Inhibitory effects of oral administration of ginsenoside Rh2 on tumor growth in nude mice bearing serous cyst adenocarcinoma of the human ovary

We examined the inhibitory effect of the oral administration of ginsenoside Rh2 (Rh2) on tumor growth in nude mice bearing human ovarian cancer cells (HRA). In the first experiment, it was revealed that daily administration of 30 microM Rh2 significantly inhibited tumor growth. In the second experiment, therefore, various concentration of Rh2 (1, 15, 30, 60, 120 microM) were administered every day for 91 days, beginning the day after tumor inoculation. Treatment with Rh2 resulted in a remarkable retardation of the HRA cell tumor growth. In particular, tumor growth in mice treated with 15, 30 and 120 microM Rh2 was significantly inhibited, compared to that in CDDP treated mice as well as in untreated mice. Consequently, 50% survival in nude mice treated with 15, 30 and 120 microM Rh2 was significantly prolonged, compared to that not only in untreated mice but also in CDDP treated mice. No side effect was observed in any mice treated with Rh2. Red ginseng containing Rh2 has been used exclusively, orally administered. In the present study, we considered that oral administration of Rh2, which is a component of red ginseng, has strong inhibitory effects on human ovarian cancer cell growth in nude mice.     

Proton MR spectroscopy and neuropsychological testing in adrenoleukodystrophy

Melatonin, the chief hormone secreted by the pineal gland, has been previously shown to inhibit human breast cancer cell growth at the physiological concentration of 1 nM in vitro. In this study, using the estrogen receptor (ER)-positive human breast tumor cell line MCF-7, we have shown that 10 microM L-buthionine-[S,R]-sulfoximine (L-BSO), an inhibitor of gamma-glutamylcysteine synthetase (the rate-limiting enzyme in glutathione synthesis), blocks the oncostatic action of 1 nM melatonin over a 5-day incubation, indicating that glutathione is required for melatonin action. The result was repeated with ZR75-1 cells, suggesting that the glutathione requirement is a general phenomenon among ER+ breast cancer cells. Addition of exogenous glutathione (1 microM) to L-BSO-treated groups restored the melatonin response in both cell lines. Further demonstration of the importance of glutathione was shown using the ER- breast tumor cell line HS578T, which is normally unresponsive to melatonin. Growth in this cell line was inhibited in the presence of 1 microM ethacrynic acid (an inhibitor of glutathione S-transferase) plus 1 nM melatonin, and this effect was blocked with 10 microM L-BSO. We also observed a steady decrease of intracellular glutathione in MCF-7 cells over a 5-day incubation, suggesting that these cells metabolize glutathione differently than do normal cells.     

Differential regulation of glial cell line-derived neurotrophic factor (GDNF) expression in human neuroblastoma and glioblastoma cell lines

Human SK-N-AS neuroblastoma and U-87MG glioblastoma cell lines were found to secrete relatively high levels of glial cell line-derived neurotrophic factor (GDNF). In response to growth factors, cytokines, and pharmacophores, the two cell lines differentially regulated GDNF release. A 24-hr exposure to tumor necrosis factor-alpha (TNFalpha; 10 ng/ml) or interleukin-1beta (IL-1,; 10 ng/ml) induced GDNF release in U-87MG cells, but repressed GDNF release from SK-N-AS cells. Fibroblast growth factors (FGF)-1, -2, and -9 (50 ng/ml), the prostaglandins PGA2, PGE2, and PGI2 (10 microM), phorbol 12,13-didecanoate (PDD; 10 nM), okadaic acid (10 nM), dexamethasone (1 microM), and vitamin D3 (1 microm) also differentially effected GDNF release from U-87MG and SK-N-AS cells. A result shared by both cell lines, was a two- to threefold increase in GDNF release by db-cAMP (1 mM), or forskolin (10 microM). In general, analysis of steady-state GDNF mRNA levels correlated with changes in extracellular GDNF levels in U-87MG cells but remained static in SK-N-AS cells. The data suggest that human GDNF synthesis/release can be regulated by numerous factors, signaling through multiple and diverse secondary messenger systems. Furthermore, we provide evidence of differential regulation of human GDNF synthesis/release in cells of glial (U-87MG) and neuronal (SK-N-AS) origin.     

2-chlorodeoxyadenosine (cladribine) induced allergic cutaneous reactions with eosinophilia in a patient with B-cell chronic lymphocytic leukemia

We present a 68-year old patient with B-cell chronic lymphocytic leukemia (CLL) treated with 2-chlorodeoxyadenosine (2-CdA). This is the first reported patient with B-CLL in whom skin lesions and eosinophilia were observed simultaneously. The most frequent side effect of this drug is myelosuppression with pancytopenia. So far, there have been few reports of cases where either skin reactions or eosinophilia, occurring separately, were observed as side effects from 2-CdA treatment.     

Allogeneic cell therapy for a murine mammary carcinoma

The effect of allogeneic cell therapy on tumor growth was studied in a murine model of mammary carcinoma (4T1) as an experimental model of solid tumors in humans. i.v. inoculation of 4T1 (H-2d) cells into syngeneic mice [BALB/c or (BALB/cXC57BL/6)F1] (F1) carrying the H-2d histocompatible antigens results in tumor colonies in the lungs that finally cause the death of all of the mice. Sublethally irradiated F1 mice were inoculated with 4T1 cells to simulate minimal residual disease and with immunocompetent splenocytes derived from naive donors of F1 (syngeneic), BALB/c (syngeneic to the tumor but semiallogeneic to the host), or C57BL/6 (allogeneic to the tumor and semiallogeneic to the host) mice. The survival of F1 tumor-bearing mice that were treated with allogeneic C57BL/6 splenocytes was significantly prolonged (P < 0.02) compared with hosts given F1 or BALB/c-derived splenocytes that are syngeneic to 4T1 tumor cells. Adoptive transfer of lung cells that were isolated from F1 primary mice inoculated with 4T1 cells and syngeneic BALB/c or F1 splenocytes led to local tumor growth and death in secondary recipients. In contrast, only 1 of 22 secondary recipients developed tumors when inoculated with lung cells derived from F1 mice given allogeneic C57BL/6 splenocytes. All of the 21 secondary hosts survived disease-free for a follow-up time of >200 days. These results indicate that immunocompetent cells allogeneic to the mammary carcinoma cells were able to inhibit tumor development in the primary hosts and to prevent tumor growth in the adoptive recipients, which suggests that allogeneic cell therapy may be an efficient antitumor tool to eradicate minimal residual disease in human solid tumors.     

Cosmetic results of breast conserving therapy for breast carcinoma. Treatment results from the Heidelberg Radiation Clinic in the years 1984 to 1992]

We examined the effect of an antioxidant and protein kinase inhibitors on prostaglandin E2 (PGE2) release from Balb/c 3T3 mouse fibroblast cells induced by quinolone phototoxicity. Simultaneous administration of sparfloxacin (SPFX) or lomefloxacin (LFLX) at 12.5 to 100 microM and ultraviolet-A (UVA) irradiation for 10 min markedly elevated PGE2 concentration in the incubation medium, whereas levofloxacin (LVFX) at concentrations up to 100 microM and UVA irradiation did not increase PGE2 concentration. Pretreatment with 100 microM pyrrolidine dithiocarbamate (PDTC), an antioxidant, or 1 microM calphostin C, a selective inhibitor of protein kinase C (PKC), completely inhibited the elevation of PGE2 in the 24-h incubation medium; pretreatment with 10 microM H7, a cyclic nucleotide-dependent protein kinase, and PKC or 1 microM herbimycin A, a tyrosine kinase inhibitor, inhibited the PGE2 elevation by 29 to 39%. Conversely, 25 nM staurosporine significantly augmented the PGE2 elevation by quinolones plus UVA. Interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) were not detected in the incubation medium of 3T3 cells after quinolone plus UVA, corresponding to the lack of effect of antibodies against IL-1alpha, IL-1beta, and TNFalpha on PGE2 release from 3T3 cells. These results suggest that PGE2 production in 3T3 cells by quinolone phototoxicity is modulated by reactive oxygen species, PKC, and tyrosine kinase, but not by IL-1 or TNFalpha.     

Anticancer efficacy in vivo and in vitro, synergy with 5-fluorouracil, and safety of recombinant methioninase

The elevated exogenous-methionine dependency of tumors for growth has been observed in all major cancer cell types. We have previously cloned a methioninase (rMETase) from Pseudomonas putida to deplete methionine. Growth inhibition followed by apoptotic cell death was induced by treatment of tumor cells with rMETase in vitro. A single i.p. injection of 300 units of rMETase can lower the serum methionine level in the mice from 70 microM to less than 1 microM within 2 h and maintain this depleted level for 8 h. Repeated dosing of rMETase of tumor-bearing mice could be administered without acute immune-hypersensitivity. rMETase treatment demonstrated growth inhibitory activity against human tumors in nude mice, including those which were multiple drug-resistant. No body weight loss or hematotoxicity, except a slight anemia, was found throughout the therapy. The combined treatment of the Lewis lung carcinoma with a fixed rMETase dose and increasing doses of 5-fluorouracil (5-FU) resulted in a dose-dependent enhanced antitumor efficacy for survival as well as tumor growth inhibition. Thus, methionine depletion by rMETase potentiates the antitumor efficacy of 5-FU. The data presented in this report thus indicate that rMETase is active alone, is synergistic in combination with 5-FU, and has negligible toxicity suggesting a novel clinical approach for effective cancer therapy.     

Induction of superoxide by 12-O-tetradecanoylphorbol-13-acetate and thapsigargin, a non-phorbol-ester-type tumor promoter, in peritoneal macrophages elicited from SENCAR and B6C3F1 mice: a permissive role for the arachidonic acid cascade in signal transduction

Local production of reactive oxygen intermediates, e.g., superoxide anion, by tumor promoter-stimulated inflammatory macrophages (MPs) may contribute significantly to tumor development in classical models of two-stage chemical-induced carcinogenesis in murine skin. In the studies reported herein, peritoneal MPs elicited from phorbol-ester-sensitive SENCAR mice demonstrated a time- and dose-dependent release of superoxide anion (4-6 nmol/10(6) cells) when stimulated by 200 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) in vitro; MP superoxide response was significantly inhibited (50-70%) by preincubation with 40 microM 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7), a protein-kinase inhibitor. Alternatively, TPA-stimulated MPs derived from relatively resistant B6C3F1 mice generated negligible superoxide under the same conditions. A similar strain-dependent induction of superoxide was observed when MPs were stimulated with thapsigargin (TG), a tumor promoter previously shown to act independently of protein kinase C (PKC). TG-stimulated SENCAR MPs released a significant amount of superoxide (2-3 nmol/10(6) cells) that was not inhibited by H-7; MPs from B6C3F1 mice demonstrated negligible stimulation by TG. Preincubation of SENCAR MPs with 100 microM dibromoacetophenone, an inhibitor of phospholipase A2, completely suppressed the superoxide induced by TPA and TG stimulation. Like TPA, 50 microM 1-oleoyl-2-acetylglycerol, a diacylglycerol analogue and PKC activator, also induced a significant amount of superoxide from SENCAR MPs only. In parallel with the superoxide findings, TPA and TG stimulated significantly greater [3H]arachidonic acid release from prelabeled SENCAR MPs (a 32% and 48% increase, respectively, over unstimulated controls) relative to MPs from B6C3F1 mice. Two-dimensional gel-electrophoretic analysis indicated that TPA-induced phosphorylation of a 47-kDa protein (a presumed substrate for PKC previously linked to NADPH oxidase activation in guinea pig and human polymorphonuclear leukocytes) occurred in MPs from both SENCAR and B6C3F1 mice. Therefore, arachidonic acid production may be a common biochemical pathway by which phorbol-ester--and non-phorbol-ester--type tumor promoters activate MPs in SENCAR mice; such a response may be "permissive" for additive (or synergistic) interactions with PKC-driven signal pathways.     

Vaccination with tumor cells engineered to secrete interleukin 2-immunoglobulin G fusion protein induces tumor rejection

Here we provide proof that the injection of tumor cells engineered to secrete interleukin 2 (IL-2)-IgG chimeric proteins locally induces potent antitumor responses, which are more effective than tumor transfection with IL-2 alone. Murine plasmacytoma cells (J558L) were stably transfected with DNA coding for a human IL-2-IgG1 or a murine IL-2-IgG2b fusion protein and were injected s.c. into syngeneic BALB/c mice. Evaluation of tumor growth and rejection patterns showed that IL-2-IgG secretion by transfected J558L tumor cells induced their rejection in all animals tested, similar to the rejection of J558L cells engineered to secrete IL-2 alone, whereas treatment with parental cells was lethal. However, mice treated with IL-2-IgG-secreting J558L cells (human IL-2-IgG1 and murine IL-2-IgG2b) exhibited a significantly stronger tumor immunity against a later challenge with parental J558L cells than mice treated with IL-2-secreting tumor cells.     

Inhibitory effects of ginsenoside Rh2 on tumor growth in nude mice bearing human ovarian cancer cells

Ginsenoside Rh2 (Rh2), isolated from an ethanol extract of the processed root of Panax ginseng CA Meyer, inhibits the growth of B16 melanoma cells. This study was designed to evaluate the ability of Rh2 to inhibit growth of human ovarian cancer cells (HRA) in vitro and in nude mouse. Rh2 inhibited proliferations of various established human ovarian cancer cell lines in a dose-dependent manner between 10 and 60 microM in vitro and induced apoptosis at around the IC50 dose. When HRA cells were inoculated s.c. into the right flank of nude mice, all mice formed a palpable tumor within 14 days. Although i.p. administration of Rh2 alone hardly inhibited the tumor growth, when Rh2 was combined with cis-diamminedichloroplatinum(II) (CDDP) the tumor growth was significantly inhibited, compared to treatment with CDDP alone. When mice were treated p.o. with Rh2 daily (but not weekly), the tumor growth was significantly (P<0.01) inhibited, compared to CDDP treatment alone. When Rh2 was combined with CDDP, the degree of tumor growth retardation was not potentiated. The survival time was significantly (P<0.05) longer than that of medium alone-treated controls or the group treated with CDDP alone. Then, we examined whether p.o. administration of Rh2 has a dose-dependent inhibitory effect on the tumor growth. I.p. and weekly administration of CDDP had more potent antitumor activity in the order of 1 mg/kg, 2 mg/kg and 4 mg/kg, whereas p.o. and daily administration of Rh, (0.4 to 1.6 mg/kg) not only had antitumor activity comparable to that of 4 mg/kg CDDP, but also resulted in a significant increase of the survival. Doses of Rh2 used in this study did not result in any adverse side-effects as confirmed by monitoring hematocrit values and body weight, unlike 4 mg/kg CDDP, which had severe side-effects. It is noteworthy that p.o. but not i.p. treatment with Rh2 resulted in induction of apoptotic cells in the tumor in addition to augmentation of the natural killer activity in spleen cells from tumor-hearing nude mice. Thus, particularly in view of the toxicity of CDDP, Rh2 alone would seem to warrant further evaluation for treatment of recurrent or refractory ovarian tumor.     

Stable expression of the recombinant human VIP1 receptor in clonal Chinese hamster ovary cells: pharmacological, functional and molecular properties

We stably transfected Chinese hamster ovary (CHO) cells with the recombinant human vasoactive intestinal peptide (VIP)1 receptor. A clone referred to as Clone 15 was isolated and studied for receptor properties. The following data were obtained: (1) one class of binding site was identified by Scatchard analysis of [125I]VIP binding to cell membranes with a Kd of 0.41 nM and a Bmax of 1.62 pmol/mg protein; (2) the constant Ki for the inhibition of [125I]VIP binding by VIP and related peptides was: VIP (0.9 nM) = pituitary adenylate cyclase-activating peptide (PACAP)-27 (1.3 nM) < PACAP-38 (6.8 nM) < helodermin (46.0 nM) < human growth hormone-releasing factor (GRF) (0.6 microM) < peptide histidine methionineamide (2.0 microM) < secretin (> 10 microM); (3) cross-linking experiments using [125I]VIP identified a single M(r) 67000 recombinant receptor; (4) VIP stimulated cAMP production in Clone 15 cells with an EC50 of 0.20 nM; (5) some previously described VIP receptor antagonists including [4-Cl-D-Phe6, Leu17]VIP, [Ac-Tyr1,D-Phe2]GRF-(1-29) amide and VIP-(10-28) inhibited [125I]VIP binding with a Ki of 0.7, 1.6 and 2.5 microM, respectively. They failed to stimulate cAMP production in Clone 15 cells and inhibited, at least partially, the VIP (0.3 nM)-evoked cAMP production; (6) exposure of Clone 15 cells to 10 nM VIP for 24 h resulted in a sharp decrease in Bmax in Clone 15 cells (0.43 vs. 1.62 pmol/mg protein in control cells) and in the potency and efficacy of VIP in stimulating cAMP. Moreover, immunofluorescence studies using confocal microscopy indicated that the receptor was internalized and sequestered in vesicular structures within the cells. It is concluded that Clone 15 cells provide a valuable tool to further characterize various functional and pharmacological aspects of the human VIP1 receptor.     

Model of implantation of tumor cells simulating recurrence in colonic anastomosis in mice

PURPOSE: Local recurrence after colorectal cancer surgery is usually perianastomotic. An experiment was designed to investigate whether free intraluminal cells can penetrate through a colonic anastomosis and thereby cause local recurrence. METHODS: BALB/c and C57/BL mice underwent ascending colotomy followed by watertight anastomosis. Thereafter, CT-26 murine colon carcinoma cells were injected into the cecal lumen 2 cm proximal to the anastomosis of syngeneic BALB/c mice, whereas B-16 murine melanoma cells were injected in the same fashion into C57/BL mice. Control animals without anastomosis received similar injections. Animals were killed 24 hours, 72 hours, and 30 days after surgery and were checked for tumorigenesis. RESULTS: Results of peritoneal fluid cytology were negative after 24 hours, whereas after 72 hours cancer cells were identified in the peritoneal fluid of 80 percent of mice with colotomy and anastomosis compared with 20 percent of control mice. Thirty days after surgery, 11.1 percent of the control BALB/c mice developed pericecal tumor growth, similar to the overall rate of murine melanoma in C57/BL. In mice with anastomoses, perianastomotic tumor growth was observed in 47.5 percent of BALB/c mice (P < 0.001) and was correlated with the number of injected cells. Tumor growth reached approximately 75 percent tumor take with high cell densities, whereas in C57/BL mice no difference was found between the experimental and control groups. CONCLUSIONS: The findings suggest that free intraluminal cancer cells of colonic origin may penetrate through watertight anastomoses and implant on the anastomotic or peritoneal surface and initiate tumor growth. This anastomotic penetration is cell-mass dependent. The reported experimental model is simple, reproducible, and advantageous for studies of colonic anastomosis.     

I1-imidazoline receptors and cholinergic outflow to the airways

The modulatory effect of bradykinin on electrically-induced noradrenaline release was assessed in isolated atria from normal and B2 knockout transgenic mice preincubated with [3H]noradrenaline. Concentrations of 1, 3 and 10 nM of bradykinin did not significantly alter the outflow of radioactivity whereas higher concentrations of bradykinin (30 and 100 nM) enhanced it. The facilitatory effect of 30 nM bradykinin was inhibited by a selective bradykinin B2 receptor antagonist. Hoe 140 (D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin, 30 nM), and by a protein kinase C inhibitor, bisindolylmaleimide (1 microM). The co-administration of bradykinin (1 to 100 nM) with either [Leu8]des-Arg9-bradykinin (100 nM), AcLys[DbetaNal7,Ile8]des-Arg9-bradykinin (30 nM) (bradykinin B1 receptor antagonists) or diclofenac (1 microM) (a cyclooxygenase inhibitor), shifted the facilitatory effect of bradykinin to lower concentrations. The facilitatory effect of bradykinin also was enhanced by enalaprilat (1 microM) and mergetpa (1 microM), inhibitors of angiotensin-converting enzyme (kininase II) and kininase I, respectively. In contrast, selective bradykinin B1 receptor agonists, des-Arg9-bradykinin (1 to 100 nM) and Sar[D-Phe8]des-Arg7-bradykinin (1 to 100 nM), did not significantly affect the stimulation-induced outflow of radioactivity. Neither bradykinin (100 nM) nor des-Arg9-bradykinin (100 nM) had any modulatory effect in B2 knockout transgenic mice. These findings suggest that the facilitatory effect of bradykinin on noradrenaline release in the mouse atria is mediated exclusively by presynaptic bradykinin B2 receptors which are linked to protein kinase C. The greater release of noradrenaline with bradykinin under inhibition of prostaglandins production and kininases I and II activity might be of importance in pharmacotherapies.