Antisense oligonucleotide intralesional therapy for human PC-3 prostate tumors carried in athymic nude mice

To address the question whether calretinin (CR) may protect cells against Ca2+ overload or trophic factor deprivation, PC12 cells were transfected with plasmids containing a CR coding region under control of a cytomegalovirus promoter. Nerve growth factor (NGF) treatment induced differentiation, increased transfection efficiency (at least 10-fold) and activated the CR gene (as found by RNase protection method and immunohistochemistry). Exogenous CR expression was identified either in living cells by fluorescence of green fluorescent protein (when the CR coding region was fused to this protein) or in fixed cells by CR immunoreactivity. Undifferentiated and NGF-differentiated populations of transfected cells were incubated in the presence of a Ca(2+)-ionophore or in media deprived of serum or NGF. Expression of exogenous CR in undifferentiated or NGF-treated cells (due to transfection) or endogenous CR (due to gene activation by NGF) did not render PC12 cells more resistant to insults such as Ca(2+)-overload and trophic factor deprivation.     

Giant cell tumor of bone with selective metastases to mediastinal lymph nodes

We examined the efficiency of disease-specific "standard" chemotherapies epirubicin, cyclophosphamide (EC); cyclophosphamide, vincristine, doxorubicin, etoposide, prednisolone (CHOEP); epirubicin, ifosfamide (EPI/IFOS) for peripheral blood progenitor cell (PBPC) mobilization in comparison to well-characterized mobilization protocols, i.e. etoposide, ifosfamide, cisplatin, epirubicin (VIPE) and dexamethasone, carmustine, etoposide, cytarabine, melphalan (DexaBEAM). Twenty-seven patients with various malignancies underwent 75 apheresis procedures for PBPC collection. Median cell yields from all 75 aphereses were 1.18 x 10(5) mononuclear cells/kg [range (0.28-3.7) x 10)8)], 1.4 x 10(5) granulocyte/macrophage-colony-forming units (CFU-GM)/kg [range (0.2-11) x 10(5)] and 3.3 x 10(6) CD34+cells/kg [range (0.35-17.7) x 10(6). CD34+/ CD90+ cells could be mobilized by all mobilization regimens used. The difference observed in the mobilization of CD34+ cells was only of low significance when the mobilization regimens were compared, whereas the mobilizations of MNC and CFU-GM were significantly different between the groups. Breast cancer patients treated with the VIPE regimen (including pretreated women) had a significantly higher CFU-GM rate than patients treated with EC (P=0.0005). Mobilized CD34+ PBPC were correlated with CFU-GM in all apheresis products. The linear correlation coefficients differed for the various mobilization groups: DexaBEAM (r=0.9, P < 0.0001), VIPE (r=0.68, P=0.0024), CHOEP (r=0.52, P=0.022), EPI/ IFOS (r=0.34, P=0.11) and EC (r=0.23, P=0.2). We conclude that clonogenic assays can provide additional information about the autotransplant quality, particularly when alternative or new mobilization regimens are being investigated.     

Inhibition of tumor cell adhesion to lymph nodes by laminin-related peptide and neuraminidase

BACKGROUND: Adhesion to lymph nodes, rather than growth stimulation, accounted for preferential colonization of lymph nodes by a metastatic B16 melanoma. We investigated these adhesive interactions. METHODS: Four classes of molecules were tested for inhibition of melanoma adhesion to cryostat sections of lymph node. RESULTS: Calcium chelators ethylenediaminetetraacetic acid and ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetra ace tic acid completely inhibited adhesion (50% adhesion, half-maximal inhibition, at 1 to 3 mmol/L). Cytochalasin B, which impairs contractile microfilaments, inhibited adhesion (60% adhesion at .001 mmol/L, 28% at .01 mmol/L). Colchicine, which disaggregates microtubules, had a similar effect (20% at .01 mmol/L, lowest dose tested). Trypsin slightly increased adhesion (125% adhesion at 10 micrograms/ml). Neuraminidase, which removed sialic acid residues, inhibited it (50% adhesion at 5 micrograms/ml). Gly-arg-gly-asp-ser, a peptide with a cell binding sequence of fibronectin, did not consistently inhibit adhesion (69% adhesion at 0.1 mg/ml, 83% adhesion at 1 mg/ml) or substantially differ from gly-arg-gly-glu-ser-pro (59% adhesion at 0.1 mg/ml, 90% adhesion at 1 mg/ml). In contrast, a peptide with a cell binding region of laminin (tyr-ile-gly-ser-arg) inhibited adhesion (50% adhesion at .05 mg/ml). CONCLUSIONS: Tumor cell-lymph node adhesion is a calcium-dependent process, requiring a functional cytoskeleton, that is mediated by both sialic acid moieties and trypsin-resistant, laminin-related, adhesion molecules.     

Identification of dendritic cells as a major source of interleukin-6 in draining lymph nodes following skin sensitization of mice

Skin sensitization with chemical allergens is associated with the activation and proliferation of T lymphocytes within lymph nodes draining the site of exposure. These events are accompanied by the secretion of interleukin-6 (IL-6) by lymph node cells (LNC). We have investigated the cellular source of IL-6 seventy-two hours following primary exposure of mice to the contact allergen oxazolone. Immunocytochemical analyses of sections of activated lymph nodes have revealed that cells expressing IL-6 are located within the T-dependent lymph node paracortex, with none present in lymphoid follicles. Cells which expressed IL-6 cofractionated exclusively with LNC of low buoyant density, the majority of which also expressed membrane Ia and had a dendritic morphology. Depletion of dendritic cells from LNC culture was associated with a significant decrease in the secretion of IL-6 by the residual population. These data demonstrate that dendritic cells are a major source of IL-6 within lymph nodes during primary immune responses to cutaneous antigens.     

Squamotacin: an annonaceous acetogenin with cytotoxic selectivity for the human prostate tumor cell line (PC-3)

A 12-year-old child with tricuspid atresia and acquired hypoplasia of the left pulmonary artery was successfully treated with unilateral Fontan operation. Angiography at age 2 months had shown a normal left pulmonary artery, and a modified Potts shunt was performed. An emergency central shunt was required a year later. Reinvestigation 5 years after the initial operation revealed severe hypoplasia of the left pulmonary artery.     

Mast cell activation and migration to lymph nodes during induction of an immune response in mice

The mast cell response in skin and lymph nodes was examined during the sensitization phase of dinitrofluorobenzene (DNFB)-induced contact hypersensitivity in mice. Degranulation of 62% of mast cells in DNFB-exposed skin was evident within 30 min of a dual application of DNFB, reaching a peak of 77% at 24 h, and persisting in 42% after 5 d. Abundant expression of macrophage inflammatory protein (MIP)-1alpha and MIP-1beta mRNAs and proteins was observed in keratinocytes, and mast cell degranulation was significantly inhibited after administration of neutralizing antibodies to MIP-1alpha, but not MIP-1beta. During DNFB sensitization, the mast cell density in the skin decreased by half, concurrent with a fivefold expansion of mast cell numbers in draining lymph nodes. Fluorescent-labeled mast cells injected into the skin appeared in draining lymph nodes after application of DNFB, followed by subsequent migration to the spleen. In lymph nodes, mast cells were an abundant and predominant source of MIP-1beta, neutralization of which partially inhibited T lymphocyte recruitment. These results indicate that mast cells contribute to the induction of this primary immune response by activation at and migration from the site of antigen encounter to draining lymph nodes, wherein they mediate T lymphocyte recruitment by production of MIP-1beta.     

Targeted cytotoxic analog of luteinizing hormone-releasing hormone AN-207 inhibits the growth of PC-82 human prostate cancer in nude mice

OBJECTIVE: To examine the effect of gender differences among older adults hospitalized for an acute myocardial infarction (AMI) on subsequent health outcomes. DESIGN: Secondary analysis of the Longitudinal Study on Aging. Data from baseline interviews (1984) and three biennial (1986, 1988, and 1990) re-interviews were linked to Medicare hospitalization and National Death Index records for 1984-1991. PARTICIPANTS: A total of 6071 community-dwelling adults aged 70 years or older at baseline. METHODS: Pooled and stratified multivariable models were used to examine gender differences in the independent effects of being hospitalized for an AMI on all-cause mortality, the risk and volume of subsequent hospitalization, and increases in the number of functional limitations. Two comparison groups were used. RESULTS: Three hundred fifty-seven AMI cases (6%; 172 women and 185 men) were compared with 3976 hospitalized controls and 1738 nonhospitalized controls. The risk of all-cause mortality for AMI cases was greater than that for either hospitalized controls or nonhospitalized controls (referent), and this increased risk was significantly (P < .001) stronger for women (adjusted hazards ratio (AHR) = 14.24, 95%CI = 10.99, 18.46) than for men (AHR = 9.91, 95%CI = 7.75, 12.67). Overall, AMI cases were also more likely to be hospitalized subsequently than the hospitalized controls (referent; adjusted odds ratio (AOR) = 1.47, 95%CI = 1.17, 1.85), although in the stratified analysis this association held for men (AOR = 1.73, 95%CI = 1.25, 2.41) but not for women (AOR = 1.25, 95%CI = .90, 1.73). Among those subsequently hospitalized, both women and men AMI cases consumed more hospital resources than the hospitalized controls, and there were gender differences suggesting that the effects on total charges and length of stay were greater for women than for men with AMI. Finally, although the AMI cases had greater adjusted mean increases in the number of instrumental activities of daily living limitations and lower body limitations than the nonhospitalized controls, they were no worse off than the hospitalized controls, and there were no gender differences in those effects. CONCLUSION: Relative to the appropriate comparison groups, hospitalization for an AMI increases the risk of death and the total costs and lengths of stay of subsequent hospitalizations for women more than for men. Therefore, increased primary prevention, diagnosis, and treatment efforts should be directed toward women.     

Dexamethasone-induced cytotoxic activity and drug resistance effects in androgen-independent prostate tumor PC-3 cells are mediated by lipocortin 1

We studied the seated buttock pressure distribution in six paraplegic patients by means of computerized pressure mapping. They were all male and their age ranged from 18 to 48 years old. Their level of paralysis varied from Th5 to L1. Five kinds of wheelchair cushions were studied: an air cushion, a contour cushion, a polyurethane foam cushion, a Cubicushion (which is made of polyurethane foams) and a silicone gel cushion. A tactile sensor consisting of 2064 matrices was used for measuring the buttock pressure distribution and the data was analyzed on a personal computer. Peak pressures measured for each cushion were as follows (in descending order): the Cubicushion, the polyurethane foam cushion, the contour cushion, the silicone gel cushion, and the air cushion. The areas of total contact measured for each cushion were as follows (in descending order): the air cushion, the silicone gel cushion, the polyurethane foam cushion, the contour cushion and the Cubicushion. Based on these findings, we conclude that the most advantageous cushion is the air cushion or the silicone gel cushion. Likewise, we conclude that the Cubicushion is not practical for pressure sore prevention.     

Skip metastasis to the mediastinal lymph nodes in non-small cell lung cancer

BACKGROUND: Whether any difference exists in clinical characteristics between resected non-small cell lung cancer with either skip or ordinary mediastinal lymph node metastases (N2 disease) needs to be clarified. METHODS: There were 110 patients with stage IIIA N2 disease. Thirty-three patients demonstrating no metastasis at the hilar nodes [skip (+) group] were compared with the other 77 patients [skip (-) group]. To investigate the extent of nodal involvement, we classified the mediastinal lymph nodes into three regions (superior, inferior, or aortic). RESULTS: There were no significant differences regarding histologic type, T status, or the site of the primary tumors between the skip (+) and the skip (-) N2 groups. In the skip (+) group, mediastinal node metastasis was found in only one region (level 1) in 30 patients (90.9%) and in two regions (level 2) in 3 (9.1%), whereas 28 patients (36.4%) from the skip (-) group revealed mediastinal metastasis at two or three regions (level 2 or 3). The overall survival rate at 5 years after operation was 35% in the skip (+) group and 12.7% in the skip (-) group (p = 0.054). This favorable clinical outcome in the skip (+) group could be explained partially by the higher proportion of patients with level 1 metastases. Furthermore, regarding patients with level 1 disease, the skip (+) group tended to have a better prognosis than the skip (-) group (p = 0.096). CONCLUSIONS: These results suggest that patients with skip mediastinal lymph node metastases represent a unique subgroup of N2 disease.     

Immunohistochemical assessment of individual tumor cells in lymph nodes of patients with non-small-cell lung cancer

PURPOSE: This prospective study was designed to evaluate the prognostic relevance and biologic characteristics of a minimal lymphatic tumor load in non-small-cell lung cancer (NSCLC). METHODS: Frozen-tissue sections from 391 regional lymph nodes of 72 patients with completely resected NSCLCs, who were staged as free of metastases (pT1-3, pN0,M0,R0) by clinical tumor staging procedures and histopathologic examinations, were studied. For tumor-cell detection, we applied the alkaline phosphatase-antialkaline phosphatase (APAAP) immunostaining technique with monoclonal antibody Ber-Ep4 against two glycoproteins of 34 and 49 kd present of the surface and cytoplasm of epithelial cells. RESULTS: Individual Ber-Ep4-positive cells were detected in 11 of 72 (15.2%) cancer patients, while positive staining was consistently absent in all sections from control nodes of 24 noncarcinoma patients. No correlation between a positive lymph node finding and either the size or differentiation grade of the primary tumor or the presence of micrometastatic tumor cells in bone marrow assessed by immunocytochemistry with antikeratin monoclonal antibody CK2 was observed. Following a median observation time of 26.0 months (range, 15 to 39), patients with lymph node micrometastases showed a significantly shorter disease-free survival duration than node-negative patients (log-rank test, P = .005). The independence of this prognostic significance was demonstrated by a multivariate analysis (Cox regression model, P = .005). CONCLUSION: Our results provide evidence that the presence of single lung carcinoma cells in lymph nodes is an independent indicator of the disseminatory capacity of an individual primary tumor. Immunohistochemical assessment of micrometastases in lymph nodes is recommended for current tumor staging in NSCLC, as it might lead to better stratification of patients for adjuvant therapy.     

Inhibition of androgen action by dehydroepiandrosterone sulfotransferase transfected in PC-3 prostate cancer cells

Age-dependent loss of androgen sensitivity of the rat liver is associated with a marked increase in dehydroepiandrosterone/hydroxysteroid sulfotransferase (rStd) activity. Sulfonated steroid hormones are known to be ineffective in binding receptor proteins. These observations suggest that intracellular androgen sulfonation can physiologically influence androgen action. We have examined the inhibitory effect of rStd on androgen action in the human prostate cancer-derived PC-3 cells transfected with the rat androgen receptor (AR) expression plasmid and two androgen-responsive promoter reporter constructs (murine mammary tumor long-terminal repeat ligated to chloramphenicol acetyltransferase (CAT) gene and rat probasin androgen response element (ARE) ligated to firefly luciferase (LUC) gene). These transfected cells were dependent on 5alpha-dihydrotestosterone (DHT) for the activation of both reporter genes and showed about a 200- and a 800-fold increase of CAT and LUC activity, respectively, at 10(-10) M DHT over the no-hormone control. Expression of the sulfonating enzyme in this cell transfection system via the rStd expression plasmid caused a dose-dependent decline in the reporter activity with approximately 90% inhibition of androgen action at a rStd:AR plasmid ratio of 100. From these results we conclude that irrespective of a high level of AR, changes in the Std expression can markedly alter the androgen sensitivity of target cells.     

Administration of recombinant interleukin 12 prevents outgrowth of tumor cells metastasizing spontaneously to lung and lymph nodes

The present study investigates the ability of recombinant interleukin 12 (rIL-12) to modulate the growth of a primary tumor as well as the outgrowth of metastatic tumor cells in an ovarian carcinoma (OV-HM) model. This aggressive tumor displayed rapid growth of the primary tumor mass, high incidence of metastases to lung and lymph nodes, and invasion from the primary s.c. site to the peritoneal cavity. Starting 12 days after s.c. tumor cell implantation, several i.p. injections of rIL-12 at 2-3 day intervals resulted in regression of growing tumors. These treated mice did not show signs of metastases or tumor recurrence at the original site. One month after tumor implantation, untreated mice did not have visible lung metastasis, but some did have palpable lymph nodes. At this stage, the primary tumors of animals without palpable lymph nodes were surgically resected. When examined 2 months later, most animals had developed lymph node and lung metastases. In contrast, rIL-12 injections after tumor resection inhibited the development of metastases in both lung and lymph nodes. This contrasted with the failure of IL-2 to prevent metastases. Even for mice already showing signs of lymph node metastases or invasion of the abdominal wall, rIL-12 administration after tumor resection prevented further invasion to the peritoneal cavity and growth of metastatic tumor cells in lung. It was somewhat surprising that the IL-12 treatment of animals after 1 month of tumor growth without resection also resulted in complete tumor regression, as well as eradication of micrometastasis that would have occurred before the treatment. Moreover, they exhibited resistance to a rechallenge with the same tumor but not with a second tumor. Thus, this tumor system provides a relevant model to clinical situations in terms of treatment of advanced tumors and metastases. These results also indicate that IL-12 can induce a curative immune response, even in the face of an aggressive micrometastasizing tumor.     

beta ig-h3: a transforming growth factor-beta-responsive gene encoding a secreted protein that inhibits cell attachment in vitro and suppresses the growth of CHO cells in nude mice

beta ig-h3 is a novel gene first discovered by differential screening of a cDNA library made from A549 human lung adenocarcinoma cells treated with transforming growth factor-beta 1 (TGF-beta 1). It encodes a 683-amino-acid protein containing a secretory signal sequence and four homologous internal domains. Here we show that treatment of several types of cells, including human melanoma cells, human mammary epithelial cells, human keratinocytes, and human fibroblasts, with TGF-beta resulted in a significant increase in beta ig-h3 RNA. A portion of the beta ig-h3 coding sequence was expressed in bacteria, and antisera against the bacterially produced protein was raised in rabbits. This antisera was used to demonstrate that several cell lines secreted a 68-kD beta IG-H3 protein after treatment with TGF-beta. Transfection of beta IG-H3 expression plasmids into Chinese hamster ovary (CHO) cells led to a marked decrease in the ability of these cells to form tumors in nude mice. The beta IG-H3 protein was purified from media conditioned by recombinant CHO cells, characterized by immunoblotting and protein sequencing and shown to function in an anti-adhesion assay in that it inhibited the attachment of A549, HeLa, and WI-38 cells to plastic in serum-free media. Sequencing of cDNA clones encoding murine beta ig-H3 indicated 90.6% conservation at the amino acid level between the murine and human proteins. Finally, the beta ig-h3 gene was localized to human chromosome 5q31, a region frequently deleted in preleukemic myelodysplasia and leukemia. The corresponding mouse beta ig-h3 gene was mapped to mouse chromosome 13 region B to C1, which confirms a region of conservation on human chromosome 5 and mouse chromosome 13. We suggest that this protein be named p68 beta ig-h3.     

Cancer metastases to the cervical lymph nodes in an unknown primary location of the tumor

Out of 120 patients hospitalized with the diagnosis of metastases to the cervical lymph nodes and unknown primary location of the tumor, tumors were detected in 29 and lymphosarcoma in 10. In 24 (82.7%) patients the tumor was located in the upper respiratory tract and gastrointestinal tract. Malignant tumors of the naso- and stomatopharynx and vestibular part of the larynx most often metastasize into the lymph nodes. Radiotherapy was administered to all 81 patients with metastases and unknown location of the primary tumor and to 10 patients with lymphosarcoma of the cervical lymph nodes. The protocol of radiotherapy took account of the site of the metastases and probable primary tumor. A radical course provides cure in 15% of cases, as shown by follow-up of at least 2 years.     

Multicellular tumor spheroids grown from pancreatic carcinoma cell lines: use as an orthotopic xenograft in athymic nude mice

The activity of renal phosphoenolpyruvate carboxykinase (PEPCK) was measured in 3-18 month old male Fischer 344 rats after alternate periods of fasting and refeeding. To compare the induction of renal PEPCK activity in response to fasting in young and old rats, 3, 6, 12 and 18 month old animals were fasted for 30 h followed by a 24 h ad libitum refeeding period to reduce PEPCK activity toward basal levels. The refeeding period was followed by a second 24 h fasting period during which the time course of PEPCK induction was monitored in the young and old animals. The first fast resulted in over a 20% increase in renal PEPCK activity in the 3 month old and slightly over a 70% increase in the 6 month old animals. In contrast, the activity did not increase significantly in the 12 or 18 month old animals during this fasting period. Therefore the induction of PEPCK in the kidney in response to fasting appears to be altered in the older animals. Refeeding for 24 h resulted in a decrease in PEPCK activity in all four age groups; therefore there was no indication of an age-related impairment in the response of renal PEPCK to refeeding. After the refeeding period, the food was removed again and the activity was measured at short intervals over the next 24 h to determine the time course of the induction in PEPCK activity. Interestingly, during the second fast, the activity of renal PEPCK was not significantly induced in either the young or the older animals. However, the activity measured in the older 18 month rats was consistently lower during the first 12 h of the second fast as compared to the activity in the 6 month old rats. In summary, the induction of PEPCK activity in the kidney is altered with age during an initial fast; in addition, PEPCK activity is not induced in either young or old rats during a second fasting period.     

Evaluation of serum neural cell adhesion molecule as a new tumor marker in small cell lung cancer

BACKGROUND: Small cell lung cancer (SCLC) is distinguished from other histologic types of lung cancer by possessing a variety of neuroendocrine properties. Neuron-specific enolase (NSE) is the most frequently elevated tumor marker for patients with SCLC at diagnosis. To assess the value of neural cell adhesion molecules (NCAM), another possible tumor marker for small cell lung cancer, NCAM was evaluated in the sera of patients with histologically confirmed SCLC in two prospective multicenter trials. METHODS: The study includes 221 patients with SCLC, normal human blood donors (n = 34), patients with benign lung disease (n = 53), and patients with non-small cell lung cancer (n = 28). NCAM was determined by means of an enzyme immunoassay, NSE by a radioimmunoassay. RESULTS: The data show the following: (1) 51% (113 of 221) of all patients with SCLC had NCAM levels higher than 20 U/ml, 34% (75 of 221) had NSE levels higher than 25 ng/ml; (2) levels of both markers significantly differ between limited and extensive disease patients; (3) patients with pathologic NCAM and NSE levels have significantly shorter survival times; (4) a positive correlation between pretreatment NSE and NCAM levels was found (n = 221, r = 0.60); and (5) a correlation between serum marker levels and clinical status was found in follow-up studies of 19 patients. CONCLUSIONS: From these data, it is concluded that NCAM is, along with NSE, a potential tumor marker for SCLC.     

Prognostic value of immunohistochemically identifiable tumor cells in lymph nodes of patients with completely resected esophageal cancer

BACKGROUND: Current methods of disease staging often fail to detect small numbers of tumor cells in lymph nodes. Metastatic relapse may arise from these few cells. METHODS: We studied 1308 lymph nodes from 68 patients with esophageal cancer without overt metastases who had undergone radical en bloc esophagectomy. A total of 399 lymph nodes obtained from 68 patients were found to be free of tumor by routine histopathological analysis and were studied further for isolated tumor cells by immunohistochemical analysis with the monoclonal anti-epithelial-cell antibody Ber-EP4. This antibody did not stain lymph nodes from 24 control patients without carcinoma. RESULTS: Of the 399 "tumor free" lymph nodes, 67 (17 percent), obtained from 42 of the 68 patients, contained Ber-EP4-positive tumor cells. Fifteen of 30 patients who were considered free of lymph-node metastases by histopathological analysis had such cells in their lymph nodes, and 5 of the 15 had small primary tumors. Ber-EP4-positive cells found in "tumor free" nodes were independently predictive of significantly reduced relapse-free survival (P=0.008) and overall survival (P=0.03). They predicted relapse both in patients without nodal metastases (P=0.01) and in those with regional lymph-node involvement (P=0.007). All 12 patients whose lymph nodes were negative on both histopathological and immunohistochemical analysis and who were available for follow-up survived without recurrence. The presence of micrometastatic tumor cells in bone marrow had no additional prognostic value. CONCLUSIONS: Immunohistochemical examination of lymph nodes may improve the pathological staging of esophageal cancer.