Induction of Sp1 in differentiating human embryonal carcinoma cells triggers transcription of the fibronectin gene.

Cells of the human embryonal carcinoma line NEC14 proliferate as densely packed clusters consisting of small, polygonal stem cells and do not express a detectable level of fibronectin (FN). Upon induction of differentiation by treatment with N,N'-hexamethylene bisacetamide (HMBA), the level of FN mRNA increased steeply within 24 h and FN began to be accumulated, along with the organization of actin filaments in the cells. The FN promoter elements required for the activation were analyzed in reference to a cluster of GC boxes by using the chloramphenicol acetyltransferase (CAT) gene fused to 5' sequential-deletion derivatives of the promoter and promoters carrying base substitutions in the GC boxes. Among four GC boxes, GC boxes 2 and 3 had the greatest effect on promoter activation, and base substitutions in these GC boxes resulted in 80% reduction in promoter activity. The pattern of DNA-protein complex formation with these GC boxes changed drastically after induction of differentiation. The extract prepared from undifferentiated NEC14 cells formed fast-migrating complexes (UnD complexes), while the extract prepared from NEC14 cells treated with HMBA for 24 h formed slow-migrating complexes containing Sp1. Both complexes were formed predominantly with GC box 2. Base substitutions within the GC boxes completely abolished the formation of both UnD and Sp1 complexes. Consistent with these changes, the Sp1 level increased steeply within 24 h. Induction of Sp1 expression in NEC14 cells effectively stimulated the promoter activity of the transfected FN promoter-CAT constructs. These results indicate that activation of the FN promoter in differentiating NEC14 cells occurs by the steep induction of Sp1, which prevents an undifferentiated cell factor from binding to the Sp1 sites.

Enhancement of tumor growth and metastases by medroxyprogesterone acetate in transplanted uterine adenocarcinoma cells of the rat.

PIP: This study describes the effects of medroxyprogesterone acetate on tumor growth and metastases in transplanted uterine adenocarcinoma cells of the rat. High-and-low-tumorigenic cloned cells of Sprague-Dawley rat uterine adenocarcinoma originally induced by 7,12-dimethylbenz (alpha) anthracene in vivo were used. Both were derived from the same parent culture. They were cultured for more than 2 years and both retained almost the same transplantability. Survival rate of cell colonies in vitro was reduced in both lines after progesterone treatment of more than 8 mcg per ml. This reduction was dose dependent. About 1 million cells suspended in .2 ml culture medium were injected sc into the interscapular region of isologous newborn rats. At 5 weeks these rats were given .5 mg medroxyprogesterone acetate twice a week for 2 weeks. At 7 weeks they were killed. High-tumorigenic cells produced growing tumors in all newborn rats. About a third of these rats died of metastases during the 7-week observation period. Tumors produced by low-tumorigenic cells grew slowly and occasionally regressed without metastases to the lung. Tumors in female rats were larger than those in males. Enhancement of tumor growth and metastases by this progesterone compound was observed in rats inoculated with low-tumorigenic cells as compared to controls. The enhancement was not significant in tumors produced by high-tumorigenic cells. The progesterone may act immunosuppressively in vivo, or make alterations in environmental conditions of the tumors.^ieng

In vivo and in vitro tests of inhibitory effect of progesterone on cell-mediated immunity in rats bearing a syngeneic uterine adenocarcinoma.

Growth inhibition or stimulation of target adenocarcinoma cells in rats sensitized with spleen cells from syngeneic tumor-bearing rats was significantly suppressed in colony inhibition assays when the spleen cells were treated in vitro with 0.8 mug progesterone or more/ml medium. In addition, when tumor-bearing rats were treated with 1 mg medroxyprogesterone acetate weekly in vivo, the inhibiting action of the spleen cells from rats with regressed tumors was also suppressed. Progesterone thus suppressed immune spleen cells in vivo and in vitro.

Characteristics of a human cell line successively transformed by Rous sarcoma virus and simian virus 40.

Human enbryo cells were successively transformed by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) and simian virus 40 (SV40) in vitro, and the double transformant HuE 13 RS was established. From this cell line the two clonal cell lines RSa and RSb were isolated. In both, presence of SV40 T antigens was demonstrated by the fluorescent antibody technique, and the presence of RSV genomes was verified in one RSb clone by focus formation after fusion with chick embryo cells. Growth of these cells was affected by dibutyryl cAMP without marked morphologic changes. Cells were extremely sensitive to the anticellular action of human leukocyte interferon.

Spontaneous occurrence of atypical hyperplasia and adenocarcinoma of the uterus in androgen-sterilized SD rats.

In SD female rats sterilized by a single injection of testosterone propionate at 2 days after birth, the spontaneous occurrence of atypical hyperplasia and adenocarcinoma of the uterus was observed for a fairly long period (greater than 2 yr). Two atypical hyperplasias and 2 adenocarcinomas were detected in 25 androgen-sterilized rats (ASR) after 500 days of age; in contrast, in 111 normal control rats no abnormal uterine proliferation was detected during a 750-day observation period. These results indicate that a persistence of both hormone imbalances and dysfunctional uteri in ASR induces abnormal uterine proliferation at a late age.

Cellular differentiation of rat uterine adenocarcinoma cells by progesterone in vitro.

Transplantable cloned HTP/Cl culture was a stable line derived from a rat uterine adenocarcinoma that was induced by 7,12-dimethylbenz(a)anthracene in vivo and did not display density-dependent inhibition of growth. This HTP/Cl culture easily adapted to grow in a culture medium containing progesterone, 8 mug/ml. As compared with HTP/Cl culture, HTP/Cl/P8 culture grown in the presence of progesterone was contact inhibited, and a cellular differentiation was observed in the tumor tissues that developed after inoculation of cells. These actions of progesterone on uterine adenocarcinoma cells were completely reversible on removal of the hormone in vitro. These results appeared to indicate that progesterone was involved in the regulation of both cellular proliferation and differentiation. The possible mechanisms of the regulation of rat uterine adenocarcinoma cells by progesterone are discussed in relation to cellular levels of cyclic adenosine 3':5'-monophosphate in vitro.

Neural rosette formation within in vitro spheroids of a clonal human teratocarcinoma cell line, PA-1/NR: role of extracellular matrix components in the morphogenesis.

From the human teratocarcinoma-derived cell line PA-1, we established a clonal line, PA-1/NR, that stably produced a distinct cellular arrangement of neural rosettes when cultured as in vitro multicellular spheroids for 3 weeks. On immunofluorescence staining and fluorescence-activated cell sorter analyses, PA-1/NR cells in monolayer expressed the neuroectoderm-associated antigens HNK-1, NC-1, and A2B5 and the neuroblastoma-associated antigens KP-NAC8 and KP-NAC10 but lacked human embryonal carcinoma antigens, SSEA-3 or K21 antigen. Here, we investigated the developmental process of rosette formation with respect to morphological features, distribution of mitotic cells, and expression of multiple lineage-related markers and extracellular matrix (ECM) components. Ultrastructural examination of these rosettes disclosed a well-defined cavity radially surrounded by wedge-shaped or pseudostratified cells, apical microvilli and junctional complexes, and basal laminae and collagen fibrils at their basal surface. In these rosettes, many proliferating cells were detected by the immunohistochemical staining of cells incorporating bromodeoxyuridine. PA-1/NR spheroids consistently displayed neuron-specific enolase, S-100 protein, and vimentin but not glial fibrillary acidic protein, neurofilament proteins, or myelin basic protein. The rosette formation accompanied a strikingly polarized and overlapped deposition of ECM components including tenascin-carrying HNK-1 epitopes, laminin, type IV collagen, heparan, and chondroitin sulfate proteoglycans. Immunoblotting analyses showed that laminin B1 and B2 chains were constitutively expressed, whereas a fully assembled form of laminin and type IV collagen appeared only after spheroid development, suggesting that these ECM components play a morphogenetically important role in rosette formation. Close similarities between these rosettes and the neural tube of humans and experimental animals in the morphogenetic process and ECM formation lead us to propose that the PA-1/NR spheroids provide an in vitro model for the study of the earliest stage of human neurogenesis.

Molecular properties of F9 embryoglycan recognized by a unique antibody in sera from patients with germ cell tumors.

Human antibody against an embryoglycan present on a mouse teratocarcinoma cell line F9 was found in sera from 16 of 29 patients with embryonal carcinoma, yolk sac tumor, immature teratoma, and choriocarcinoma of gonadal and extragonadal origins by Farr assay. In contrast, none of the sera from patients (77 cases) with dysgerminoma, seminoma, germinoma, and mature teratoma or from patients (118 cases) with nongerm cell types of ovarian tumors contained this antibody. The antigenic embryoglycan was of high molecular weight (Mr greater than 70,000) on Sephacryl S300 column chromatography and carried binding sites for Grifonia simplicifolia agglutinin-1. The antigenic embryoglycan was also found in F9 cell-cultured medium. Absorption of patients' sera with synthetic Blood Group B trisaccharides failed to remove the antibody against F9 embryoglycan. None of these patients' sera showed higher hemagglutination titer to rabbit erythrocytes than the normal range. In contrast, alpha-galactosyl carbohydrates obtained from Ehrlich ascites tumor cells effectively inhibited the binding of patients' sera with F9 embryoglycan. These results indicate that the human antibody against F9 embryoglycan recognizes alpha-galactosyl structures that are distinct from B blood group antigen, but are cross-reactive with alpha-galactosyl structures on Ehrlich ascites cells.

Limitation of differential expression of HLA-A,B,C antigens on choriocarcinoma cell lines by messenger RNA for HLA heavy chain but not by beta 2-microglobulin.

The relative amounts of HLA-A,B,C antigens, beta 2-microglobulin (beta 2m), and trophoblast antigens (Trop-1 and Trop-2) were determined on nine choriocarcinoma cell lines including seven lines of gestational origin and two lines of nongestational origin (from ovary and stomach) by quantitative immunofluorescence analysis using a fluorescence-activated cell sorter. Most of these lines expressed surface HLA to variable extents, but one had none detectable. However, all lines secreted readily measurable amounts of beta 2m. We analyzed total RNA extracted from these lines using northern blot molecular hybridization with HLA-A,B,C- and beta 2m-specific complementary DNA probes. We found no messenger RNA species which hybridized with the HLA probe in cells with no detectable HLA surface antigen and only small amounts of HLA-specific RNA in cells with low levels of HLA membrane antigen. Cells exhibiting surface HLA levels greater than about 30% of that on lymphocytes had much higher amounts of HLA-specific RNA than did choriocarcinoma cells with no or low HLA antigen expression. In contrast, RNA hybridizing with beta 2m-specific probes was present at the 20% level or higher (relative to lymphocytes) in all the cell lines tested. Thus, the expression of HLA-A,B,C is apparently limited in choriocarcinoma cells by the level of HLA heavy-chain RNA and not by the level of beta 2m RNA. We discuss these findings in relation to the normal trophoblastic or other origins of this tumor type and with respect to the regulation and function of HLA in trophoblasts.

In vivo and in vitro studies of experimental ovarian adenocarcinoma in rats.

When 7,12-dimethylbenz[a]anthracene-impregnated sutures were directly applied to the ovarian parenchyma of 8-week-old Sprague-Dawley rats (the clipping method), adenocarcinomas developed in 29 (39%) of the 75 rats during the 50-week observation period. When 20-methylcholanthrene was used, adenocarcinomas developed only in 1 (3%) of the 31 rats. Thus, the clipping method using 7,12-dimethylbenz[a]anthracene is satisfactory as an animal model of ovarian adenocarcinoma which comprises 85 to 90% of human malignant ovarian tumors. On the other hand, attempts were made to isolate cloned cell lines from these experimental ovarian adenocarcinomas in vitro, and two cloned cell lines were obtained. They were epithelioid and produced undifferentiated adenocarcinomas by back-transplantation into isologous newborn rats.

Protein phosphorylation required for the formation of E2F complexes regulates N-myc transcription during differentiation of human embryonal carcinoma cells.

The human embryonal carcinoma (EC) cell line NEC14 can be induced to differentiate morphologically by the addition of 10(-2) M N,N'-hexamethylene-bis-acetamide (HMBA). The N-myc gene is expressed at a high level in the undifferentiated cells, but the level decreased steeply after 12-24 h HMBA treatment, returning to its original level after 48 h. The alteration in the N-myc level was well correlated with the formation of complexes with the E2F motif in the N-myc promoter region, and no complex was formed with cell extracts prepared from cells treated with HMBA for 12-24 h. The absence of E2F complexes during this period was caused by an inhibitor generated by a phosphatase reaction. Treatment of the 12-h extract with a cyclic AMP-dependent protein kinase resulted in the formation of E2F complexes, and treatment of the undifferentiated (0 h) and 48-h extracts with a calf intestinal phosphatase abolished complex formation completely. An inhibitor generated by the 0-h extract after treatment with a phosphatase inhibited E2F complex formation by the untreated 0-h extract in the presence of phosphatase inhibitors, okadaic acid and sodium vanadate. One of the two E2F complexes in the undifferentiated cells contained cyclin A, but the complex with similar mobility, formed after the transient decrease in the N-myc level, did not.

A novel E2F binding protein with Myc-type HLH motif stimulates E2F-dependent transcription by forming a heterodimer.

The human embryonal carcinoma cells NEC14 can be induced to differentiate morphologically by the addition of 10(-2) M N, N'-hexamethylene-bis-acetamide and cease to grow in several days. Transcription factors of the E2F/DP family have been shown to be closely related to the regulation of cell proliferation. To analyse cellular proteins which interact with E2F in NEC14 cells, cDNA clones encoding E2F binding proteins were isolated from a lambdaZAP II NEC14 cell library with the 32P-labeled GST (Glutathione S-transferase)-E2F-1 fusion protein as a probe. One of the clones encodes E2FBP1 which has the helix-loop-helix (HLH) motif, but lacks the basic domain and the zipper structure usually found at N- and C-terminal sides to the HLH motif, respectively. The arrangement of amino acids in the helix 1 and helix 2 regions is quite similar to those of Mxi and Mad, but different from those of E2F-1 and DP-1. Western blot analysis of the immunoprecipitates prepared with anti-E2FBP1 antibody showed that E2FBP1 associates with both E2F-1 and DP-1 in vivo. E2FBP1 alone has no DNA binding activity, but bind to the E2F site through heterodimerization with E2F-1 but not with DP-1. Although E2FBP1 lacks the transactivation domain, it stimulates E2F site-dependent transcription in cooperation with E2F-1.

2',5'-oligoadenylate synthetase gene in chicken: gene structure, distribution of alleles and their expression.

We have cloned the gene for chicken 2',5'-oligoadenylate synthetase (ChOAS) by the method of polymerase chain reaction with use of ChOAS cDNA sequence. The ChOAS gene is composed of five introns and six exons containing all of the sequence of the ChOAS cDNA from the start to the stop codon. The first five exons of ChOAS gene which encode the OAS catalytic domain have a similar structure to HuOAS1 gene including the exon-intron boundaries. However, the length of introns of ChOAS gene is only 1/7 of those of HuOAS1 gene. The sixth exon of the ChOAS gene encodes the ubiquitin-like (UbL) domain of two consecutive sequence (UbL1 and UbL2) homologous to ubiquitin. ChOAS encoded in a single copy gene has at least two alleles, OAS(*)A and OAS(*)B. The differences between these two alleles are in the sixth exon of the gene; a 96-nucleotide sequence in the UbL1 portion of OAS(*)A is deleted from OAS(*)B. No OAS(*)B gene was detected in nine lines of chickens tested other than Leghorns. Almost the same levels of ChOAS-A and -B proteins induced physiologically in erythrocytes were detected in infant chickens (2-week-old), but in grown-up chickens (6-month-old) the level of erythrocyte OAS-B was markedly reduced in most of B/B chickens. Thus, the UbL domain of ChOAS is responsible for the maintenance of the OAS level in the tissue.

Suppression of late phase enhanced vascular permeability in rats by selective depletion of neutrophils with a monoclonal antibody.

We investigated the role of neutrophils in increased vascular permeability by a selective reduction of neutrophils using a monoclonal antibody, RP-3. An intraperitoneal injection of RP-3 not only selectively depleted peripheral blood neutrophils, but prevented the neutrophil infiltration to the tissues. Proteose peptone, zymosan, and BCG induced three different types of inflammatory edema, showing the early phase only, early plus late phase, and the late phase only, respectively. Only the late phase response of zymosan and BCG was inhibited by a depletion of neutrophils by RP-3, though the early phase response induced by proteose peptone and zymosan was not affected. Reconstitution of neutrophil-depleted rats by in situ injection of these cells restored the inflammatory edema induced by BCG, depending upon the number of neutrophils injected.

Selective depletion of rat neutrophils by in vivo administration of a monoclonal antibody.

A monoclonal antibody (RP-3) that depletes rat neutrophils selectively in vivo was developed by hybridization of mouse myeloma cells (P3-X63.Ag8.653) and spleen cells of BALB/c mice sensitized with peritoneal neutrophils of WKA/Hok rats. RP-3 reacted with rat neutrophils but not with lymphocytes, macrophages, natural killer cells, basophils, eosinophils, or tissues of various organs. The mitogenic responsiveness to concanavalin A (ConA), phytohemagglutinin (PHA), and lipopolysaccharide (LPS) of rats given RP-3 was not significantly different from that of normal rats. Administration of RP-3 into the peritoneal cavity of rats that had been kept under specific pathogen-free (SPF) or clean conditions induced selective depletion of circulating neutrophils to under 100/mm3 (0.5% WBC). The numbers of monocytes, lymphocytes, and platelets were not changed. Administration of 2 ml of RP-3 reduced blood neutrophils to under 100/mm3 for approximately 24 h, and administration of 1 ml caused depletion for approximately 12 h.

Interaction of nuclear factors with the regulatory region of the N-myc gene during differentiation of human embryonal carcinoma cells.

The human embryonal carcinoma cell line, NEC14, can be induced to differentiate by the addition of 10(-2) M N,N'-hexamethylene-bis-acetamide (HMBA). During the early stage of HMBA-induced differentiation, the level of N-myc expression decreased steeply and transiently, and then quickly returned to its original level after reaching a minimal level at 18 h after addition of HMBA. Nuclear run-on experiments indicated that this transient decrease is regulated at the transcription start point. To investigate the mechanism of this down-regulation, the 5'-flanking region of the human N-myc gene was cloned and sequenced. Computer analysis of the sequence revealed high homology with the 5'-flanking region of the mouse N-myc gene, especially (greater than 80%) in the region of nt positions -1777 to -1732, nt positions -763 to -501 and nt positions -260 to + 1. The patterns of protein binding to the upstream region during the early stage of NEC14 cell differentiation were analyzed by gel retardation assay. The DNA fragments VIII and X, containing the sequences of nt positions -1437 to -1237 and nt positions -1863 to -1710, respectively, formed the DNA-protein complexes which were greatly reduced in quantity in the cell extract prepared 18 h after the addition of HMBA. This reduction, however, was not observed with an extract similarly prepared from the NEC14 derivative cell line, H10, expressing the N-myc gene constitutively. These results suggest a causal connection between the complex formation and the high-level transcription of the N-myc gene.

Classification of antiarrhythmic drugs based on ventricular fibrillation threshold.

The antifibrillatory action of antiarrhythmic drugs, classified on the basis of their effects on ventricular fibrillation threshold (VFT), was investigated. The relation between drug action and cardiac excitability, orthodromic/antidromic conduction through Purkinje fibers and ventricular muscle and the restitution of premature action potential duration was studied. Drug classifications were: group A, VFT markedly increased; group B, VFT moderately increased; and group C, no significant change. Group A was subdivided according to presence or absence of the dip phenomenon and supernormal period in the anodal strength-interval curve. Drugs in group A significantly reduced the difference between effective refractory period of orthodromic and antidromic conduction and the range over which the premature action potential duration reappeared. In groups B and C, the effective refractory period in orthodromic conduction was longer than that in controls, and the range of the restitution of premature action potential duration for Purkinje fibers was reduced only slightly.

Enhanced oncogenicity of human papillomavirus type 16 (HPV16) variants in Japanese population.

To investigate whether HPV16 E6 variants carry an elevated risk for cervical cancer in Japanese population, we investigated the E6 sequence variation in 40 cervical intraepithelial neoplasias (CINs) I-III and 43 invasive cervical cancers (ICCs), all positive for HPV16. HPV16 E6 variants were frequently found in ICCs than in CINs (88 vs. 65%, P=0.01). The E6 D25E, a rare variant in Western countries, was most frequently observed in ICC (44%). CIN I/II lesions with HPV16 variants were less likely to regress than those with HPV16 prototype (P=0.048). The finding that HPV16 E6 variants represent a significant risk factor is common between Western and Japanese women despite the different distribution of each variant.

Analysis of E6 variants of human papillomavirus type 33, 52 and 58 in Japanese women with cervical intraepithelial neoplasia/cervical cancer in relation to their oncogenic potential.

The variation of the E6 region of human papillomavirus type 16 (HPV16) is associated with a high risk for cervical carcinogenesis. To see whether the same is the case with HPV33, 52 and 58, known to have high homology with HPV16, we analyzed the E6 sequence variation of these HPVs in 107 Japanese women with cervical intraepithelial neoplasia (CIN) or invasive cervical cancer (ICC): 20 HPV33-positive, 46 HPV52-positive and 41 HPV58-positive cases. HPV33 variants were more frequently observed in CINs I/II than in CIN III/ICCs (71% (5/7) versus 15% (2/13), P=0.02). In HPV52-positive cases, a single E6 variant was detected in 98% of the cases, whereas the prototype accounted for 98% of HPV58-positive cases. In summary, the distribution of E6 variants is different among HPV types tested, suggesting a link between E6 variation and oncogenic potential being type-specific.

Alternative splicing of human papillomavirus type-16 e6-messenger RNA in mouse and monkey cell-lines.

Previous studies have indicated that the splice patterns of E6-transcripts of human papillomavirus type-16 (HPV-16) are uniform. The splice ratios of E6-transcripts, however, seem to be variable in several HPV-positive cell lines, suggesting that cellular factors may affect the alternative splicing of E6-transcripts. To test this hypothesis, the splice ratios of E6-transcripts in various HPV-16 E6-expressing cell lines derived from CV-1 and 10T1/2 cells were quantitatively evaluated by S1 nuclease protection assays. The splice ratios varied among cell lines derived from the same parental lines, indicating that factors specific to cell-type do not play a major role in alternative splicing. The splice ratios appeared to be low in cell lines which prominently expressed longer than expected E6-transcripts indicating that the structure of the E6-transcript affects its splicing. Analysis of the expression patterns of COS-1 cells which transiently expressed various E6-transcript constructs suggested that structure was a factor in determining alternative splicing.