bluuebunny/crossref_metadata_2025 huggingface dataset:
Preparation and characterization of antibodies to o‐phosphotyrosine and their use for identification of phosphotyrosine‐containing proteins
Authors: Ohtsuka Masahiro , Ihara Seiji , Ogawa Ryoichi , Watanabe Toshiteru , Watanabe Yasushi
Published: February 2007

To facilitate the identification of phosphotyrosine (Ptyr)‐containing proteins, rabbit polyclonal antibodies and mouse monoclonal antibody specifically reactive to P‐tyr were prepared by hyperimmunizing the animals with P‐tyr‐conjugated bovine serum albumin or poly‐L‐lysine. As determined by a solid‐phase radioimmunoassay and an enzyme‐linked immunosorbent assay, the antibodies reacted with P‐tyr‐conjugated target antigens but not with those conjugated with phosphoserine (P‐ser) or phosphothreonine (P‐thr). This immune reaction was strongly blocked by 2 mM P‐tyr and phenylphosphate but not by P‐ser or P‐thr. The antibodies were capable of isolating, as the major P‐tyr‐containing components, a 170kd protein (most likely the EGF receptor) from EGF‐stimulated, 32P‐labelled A431 cells, and 130kd and 60kd proteins from Rous sarcoma virus (RSV)‐transformed chick cell lysate which had been labelled in vitro, with γ‐32P‐ATP. Immunofluorescent staining of RSV‐transformed cells and A431 cells showed specific localization of P‐tyr‐containing proteins in the cytoplasm, plasma membrane, and nucleoluslike structures. The results demonstrated the usefulness of the antibodies for identification or isolation of P‐tyr‐containing proteins.
Monoclonal antibody against tumour‐derived cell line shows heterogeneity of altered hepatocytes during afb1‐induced carcinogenesis
Authors: Manson M. M. , Green J. A. , Neal G. E.
Published: February 2007

Monoclonal antibodies have been raised against a tumour cell line (JBI) derived from an aflatoxin B1‐fed rat liver. One antibody MRC TU/Jl has been characterized in terms of its immunohistochemical staining in sections of control and AFB,‐fed liver and by the proteins which it immunoprecipitated. The antibody reacted with bile duct epithelium in all sections. In control liver sections there was staining of hepatocyte membranes, with increased stain in periportal regions in animals ⩾12 weeks old. In AFB,‐fed liver sections, areas of biliary hyperplasia were stained and after 4 weeks on AFB1‐diet, foci of altered hepatocytes were visible. These were negative, positive or heterogeneous with respect to antibody stain. The antibody reacted much more strongly with adenomatous and cholangiomatous tissue compared with trabecular regions of hepatic tumours which developed in animals after 14 weeks on AFB, diet and 9 months on control diet. After immunoprecipitation of 14C‐leucine‐labelled proteins from a JBI cell lysate by MRC TU/Jl, 5 bands in the molecular weight range 43,000‐49,000 daltons were visualized on 10% SDS‐PAGE gels. A similar result was obtained with 14C‐glucosamine in place of leucine, indicating that all 5 bands were glycoproteins. Deglycosylation of JBI cell lysates with CNBH1‐/IO4‐ prevented immunoprecipitation of all bands, suggesting that MRC TU/JI recognized antigenic sites on the carbohydrate portions of the molecules.
High‐ and low‐leukemogenic variants of the radiation leukemia virus (RadLV): Immunogenic, suppressive and genetic properties in relation to leukemogenic activity
Authors: Yefenof Eitan , David Ya'acob Ben , Kotler Moshe
Published: February 2007

C57BL/6 (B6) mice inoculated with the highly leukemogenic variant of the radiation leukemia virus (A‐RadLV) develop suppressor cells capable of abrogating potential anti‐tumor immunity in vitro and in vivo, Inoculation of B6 animals with the low‐leukemogenic D‐RadLV variant does not result in suppressor cell generation but induces antitumor reactive lymphocytes. A‐RadLV and D‐RadLV are not leukemogenic in BALB/c or (B6 × BALB/c)F1 (F1) mice, and reactive but not suppressor lymphocytes could be demonstrated in F1 animals inoculated with either virus. Infectivity assays and fingerprint analysis revealed that A‐RadLV and D‐RadLV contain viruses with N and B tropism. In addition, thymoma cells induced by A‐RadLV produced another virus with a fingerprint pattern containing X‐MuLV elements. The possible implications of the different virus types on the immunogenic and leukemogenic properties of the RadLV variants are discussed.
Tumor‐associated macrophages stimulate the proliferation of murine tumor cells surviving treatment with the oncolytic cyclophosphamide analogue ASTA Z‐7557: In vivo implications
Authors: Evans Robert , Duffy Theodore , Cullen Richard T.
Published: February 2007

This report is concerned with the hypothesis that tumor‐associated macrophages (TAM) present during cyclophos‐phamide (CY)‐induced tumor regression stimulate proliferation of tumor stem cells that remain after the oncolytic action of CY has dissipated. In preliminary studies, plasma or serum from CY‐injected mice was used as a source of CY metabolites. However, because of the relatively high background toxicity of normal mouse plasma/serum and the lack of a precise technique to quantify how much metabolite was present at any one time, this approach was abandoned. In place of the plasma metabolites, we used the CY analogue, 4‐(2‐sulfonatoethylthio)‐cyclophosphamide cyclohexylamine salt, Asta Z‐7557. On solubilization in water, Asta Z‐7557 yields phosphoramide mustard, one of the oncolytic metabolites of CY in vivo. This approach enabled us to quantitate the amount of drug used in vitro and to control the amount to which tumor cells and macrophages were exposed. It was demonstrated that when two different C57BL/6J tumor cell targets, the MCA/76‐9 sarcoma and the EL4 lymphoma subline, E2G.2, were treated with defined quantities of Asta Z‐7557, those cells surviving the toxic effects were stimulated to proliferate in the presence of TAM isolated from the MCA/76‐9 sarcoma. Although pretreatment of the TAM with the drug slightly diminished the stimulatory effect, this was still significantly greater than the effect of culture medium alone. In no instance was there evidence that drug treatment rendered the macrophages cytotoxic towards the tumor cells. The data supported the notion that in vivo the presence of a high proportion of TAM during CY‐induced temporary tumor regression may contribute directly to the resurgence of tumor growth.
Relationship between serum and histochemical markers for hepatitis B virus and rate of viral integration in hepatocellular carcinomas in Japan
Authors: Hino Okio , Kitagawa Tomoyuki , Sugano Haruo
Published: February 2007

The relationship between serological and histochemical markers of hepatitis B virus (HBV) and viral integration in hepatocellular carcinomas (HCCs) in Japan was investigated. Special attention was paid to the exclusion of false‐positive results due to bacterial contamination of autopsy materials. Of 85 patients with HCC, 23 were positive for serum HBsAg, 18 were positive for serum HBV antibodies and the remaining 44 were negative for serum HBV markers. Among the 23 HCCs from HBsAg carriers, 19 (82.6%) had integrated HBV DNA in the tumor DNA. In contrast, of the 18 HCCs from HBV antibody‐positive patients, only one was positive for HBV DNA integration; this particular HCC was from a longterm HBsAg carrier who became a non‐carrier by seroconversion at a later age. Of 44 HCCs from patients negative for serum HBV markers, 3 had integrated HBV DNA. Of these 3 cases, 2 were, however, histochemically positive for HBsAg in non‐cancerous portions of the liver. Thus, our present results show a high integration rate of HBV DNA in HCCs of carrier patients and an extremely low rate of viral integration in HCCs of noncarrier patients, The latter finding contrasts strongly with previous results gained from analysing materials from patients in Europe and Africa. No histological differences were apparent between HCCs with or without viral integration.
Murine monoclonal antibody LAM2 defines cell membrane determinant with preferential expression on human lung small‐cell carcinoma and squamous‐cell carcinomas
Authors: Stahel Rolf A. , Speak Jonathan A. , Bernal Samuel D.
Published: February 2007

Murine monoclonal antibodies (MAbs) were generated against a human undifferentiated lung carcinoma cell line. The hybridoma designated LAM2 produced an IgM kappa MAb with reactivity to the cell membrane. Indirect immunofluorescence staining and radioimmunoassays showed LAM2 antibody to react preferentially with lung small‐cell carcinoma (SCC) cell lines and squamous‐cell carcinoma (SQC) cell lines. LAM2 antibody also stained primary cultures of normal bronchial epithelial cells, but was unreactive with human erythrocytes and nucleated marrow cells. Indirect immunofluorescent staining with LAM2 antibody was performed on frozen sections of human tumor tissues and normal tissues. LAM2 antibody stained all 8 SCC carcinomas. 4 of 5 SQC of the lung and head and neck region, and 2 of 4 lung large‐cell carcinomas. No staining was seen on lung adenocarcinomas, breast carcinomas, ovarian carcinomas, renal cell carcinomas, colon carcinomas, or mesotheliomas. Staining was present on sections of normal bronchus, but not on normal lung parenchyma, liver, kidney, adrenal or skin. While LAM2 antibody was highly reactive with all SCC examined, its antigenic determinant was not expressed in other cell lines and tumors of presumed neuroectodermal origin, including neuroblastoma, melanoma, and bronchial carcinoid. Radioimmunoprecipitation showed the antigen defined by LAM2 antibody to have two major bands of approximate molecular weights of 45,000 and 125,000. The selective reactivity of LAM2 antibody with SCC and SQC, but not with most other tumor tissues and normal tissues, makes it a good candidate for use in clinical diagnosis and possibly serotherapy.
A prospective study of association of herpes simplex virus and human papillomavirus infection with cervical neoplasia in women exposed to diethylstilbestrol in utero
Authors: Adam Ervin , Kaufman Raymond H. , Adler‐Storthz Karen , Melnick Joseph L. , Dreesman Gordon R.
Published: February 2007

One thousand one hundred thirty‐four women, enrolled in a prospective study of the development of cervical neoplasia in women exposed to diethylstilbestrol (DES) in utero, were examined at least once a year. Twenty‐three women developed cervical intraepithelial neoplasia (CIN) of different degrees during the 7‐year follow‐up period and were matched with women who did not develop CIN during the study period. Serum samples were obtained at the time of entry into the study and at the time of diagnosis of neoplasia. In the control group, a second serum sample was obtained close to the time when CIN was diagnosed in the other group of women. The sera were studied for antibodies to both types of herpes simplex virus (HSV) by microneutralization assay and by a radioimmunoassay using, as antigens, glycoproteins VPI23 for HSV type I and VPII9 for HSV type 2. Women who developed CIN had, in the enrolment specimen which preceded the neoplasia, a higher rate of antibodies to HSV‐1 than did the matched controls (22%), while the two groups did not differ in frequency of antibodies to HSV‐2 (9%). Similar differences were observed in the samples drawn at the time of diagnosis of CIN. Women who later developed CIN and had, at the time of entry into the study, cervical epithelial changes associated with DES exposure, were infected with HSV‐1 at a higher rate than women who remained free of neoplasia or women with CIN but without DES‐associated cervical epithelial changes. Morphologic evidence of associated papillomavirus (HPV) infection was observed in 74% of CIN cases and no such observation was made in biopsy specimens taken prior to development of CIN. Morphologic evidence of HPV infection was found in a woman of the control group having squamous metaplasia. HPV structural antigens were found in 3 of 10 CIN I and CIN II cases, and none in the CIN III specimens. The possible role of the HSV and HPV infection in the pathogenesis of cervical neoplasia is discussed.
A re‐examination of the epstein‐barr virus carrier state in healthy seropositive individuals
Authors: Yao Q. Y. , Rickinson A. B. , Epstein M. A.
Published: February 2007

A panel of 24 healthy seropositive donors have been followed prospectively over a period of 15 months and monitored (1) for the level of EB virus shedding in the throat by means of a sensitive cord‐blood transformation assay; (2) for the level of virus‐infected B cells in the blood via a new in vitro protocol where “spontaneous transformation” can be seen to titrate against input cell number; (3) for anti‐EB viral antibody titres and (4) for the prevailing level of virus‐specific memory T cells in the circulation. Six donors shed easily detectable levels of EB virus into throat washings on every occasion of testing, whilst 16 other donors shed lower levels of virus detectable in throat washings on a majority (10 donors) or on a minority (6 donors) of test occasions; only 2/24 donors gave no evidence of virus shedding at any time. There was a direct relationship between the EB virus shedder status of an individual (i. e., the level of virus replication in the pharynx) and the number of infected B cells present in the circulation. These results indicate that chronic, usually low‐grade, replication of the virus at some permissive site in the oro‐and/or naso‐pharynx is very often a stable accompaniment of the asymptomatic EB virus carrier state, and may indeed be essential for the long‐term maintenance of that state.
HTLV‐positive and ‐negative T‐cell lymphomas. Morphological and immunohistochemical differences between european and HTLV‐positive japanese T‐cell lymphomas
Authors: Lennet Karl , Kikuchi Masahiro , Sato Eiichi , Suchi Taizan , Stansfeld Alfred G. , Feller Alfred C. , Hansmann Martin‐Leo , Müller‐Hermelink H. Konrad , Gödde‐Salz Elisabeth
Published: February 2007

A total of 56 cases of malignant lymphoma presumed to be of peripheral T‐cell origin were investigated with regard to histological and immunohistochemical features. The goal of the study was to determine whether virus‐associated T‐cell lymphomas can be morphologically or immunohistochemically distinguished from presumably virus‐negative T‐cell lymphomas. The cases came from endemic and nonendemic regions of Japan, the United Kingdom (including 4 Caribbean cases) and the Federal Republic of Germany. Sera of all Japanese and Caribbean patients and 8 German patients were tested for antibodies to adult T‐cell leukaemia virus‐associated antigen HTLV‐A. In all cases sections were examined blind by 5 welltrained histopathologists. In most cases cryostat sections could be prepared from fresh tissue specimens and stained with a large panel of monoclonal antibodies.

All HTLV‐A‐positive cases were morphologically classifiable as the pleomorphic type of T‐cell lymphoma. Approximately 70% of the tested cases of pleomorphic T‐cell lymphoma, however, showed a positive serum reaction for HTLV‐A. All other types of peripheral T‐cell lymphoma (T‐immunoblastic lymphoma, chronic lymphocytic leukaemia of T type, T‐zone lymphoma, “AILD type” and lymphoepithelioid cell lymphoma) were HTLV‐A‐negative and mostly observed in European patients. Thus virus‐associated T‐cell lymphomas appear to be invariably of the pleomorphic type; but pleomorphism is not specific to HTLV‐A‐positive cases. This was also evident from the results of an experiment in which 2 Japanese histopathologists attempted to recognize HTLV‐A positivity in a blind study of pleomorphic T‐cell lymphomas. A maximum of about 80% of cases were correctly identified, with about 10% false‐positive diagnoses (in HTLV‐A‐negative or presumably negative cases) and 10% false‐negative diagnoses.

The immunohistochemical analysis revealed not only many common features but also 2 distinct differences between HTLV‐A‐positive and ‐negative T‐cell lymphomas. All but one of the HTLV‐A‐positive cases showed reactivity with anti‐Tac and all cases in the virus‐positive group were negative for TÜ14. All other cases were Tac‐negative and approximately 65% of these cases exhibited reactivity with TÜ14. Preliminary cytogenetic observations suggest that there are also differences in specific chromosome aberrations.
Structural characterization and biosynthesis of gp87, a melanoma‐associated oncofetal antigen defined by monoclonal antibody 140.240
Authors: Khosravi Mohammed J. , Dent Peter B. , Liao Shuen‐Kuei
Published: February 2007

The monocolnal antibody 140.240, which reacts with a melanoma‐associated oncofetal antigen, identifies an epitope present on an 87 kd molecule present on the surface of melanoma cells. This molecule is a singlechain monomer, which by tunicamycin treatment and two‐dimensional tryptic mapping has been shown to arise from a 77 kd polypeptide precursor (p77). This precursor is rapidly glycosylated to an 83 kd intermediate (gp83) which in turn is rapidly further glycosylated into the mature 87 kd glycopolypeptie (gp87). Neuraminidase treatment of the glycopeptides indicates that only gp87 contains accessible sialic acid moieties.
A study of angiogenesis factors from five different sources using a radioimmunoassay
Authors: Shahabuddin S. , Kumar S. , West D. , Arnold F.
Published: February 2007

The use of a radioimmunoassay (RIA) demonstrated that angiogenic factors from human and animal tumours, normal bovine retinas, myocardial infarcts, synovial fluid from patients with joint diseases and wound fluid shared common antigenic determinants. Values for angiogenesis factors expressed as μg Walker tumour TAF/mg protein varied from 0.8 (wound fluid) to 207 (myocardial infarct). Activated macrophages produced an angiogenic factor which did not crossreact. Normal tissue extracts which were non‐angiogenic by the chicken chorioallantoic membrane assay also failed to cross‐react in the RIA. The antigenic similarity of angiogenic factors from such a wide variety of sources suggests that, in order to minimize therapeutic side effects, it would be best to use angiogenic agonists and antagonists locally.
The use of hybridoma cells as a murine model to study tumor immunity
Authors: Ezaki Taichi , Marbrook John
Published: February 2007

The secretion of antibody by hybridoma cells allows the growth of individual cells to be measured by a hemolytic plaque assay. Similarly, plaque reduction assays are sensitive indicators of tumor immunity. A series of strains of hybridoma have been isolated from an original isolate which is sensitive to cytotoxic T cells and NK cells. In the derivation of a spleen‐seeking variant, the cells appear to have lost immunogenic but not antigenic characteristics. This model lends itself to the quantitative study of immune constraints on tumor growth in vivo. Combined with the selection of tumor variants, the model is of wide application to many fields of tumor biology, particularly where sensitive measurement of tumor cell number and viability is crucial.
Heterogeneity in surface antigen of cell lines and clones from a murine carcinoma
Authors: Kiger Nicole
Published: February 2007

Cell lines of the murine Lewis lung tumor (3LL) were established from primary tumor site (PT) and from lung metastatic foci (Met). Expression of histocompatibility antigens was assessed by immunofluorescence with an anti‐H‐2b antibody and by tumorigenicity of the cells in different strains of mice. Analysis for tumor‐associated transplantation antigens (TATA) were performed by challenging mice with living tumor cells after pretreatment with irradiated cells or by in vivo tumor neutralization assays with different tumor cells as target cells and T lymphocytes from immunized animals as effector cells. The 3LL tumor was found to be heterogeneous in respect to tumor growth rate in vivo, metastatic capacity in vivo, expression of normal histocompatibility antigens and expression of “TATA”. Moreover, monoclonal antibody B1(H11‐115) binding to a Met cell surface antigen, but not to normal cells, was obtained. The antibody bound to 3LL tumor cell lines from primary tumor (PTH, PTV), from a mixture of metastatic foci (MetH, MetV), from different metastatic foci originating from 3 different mice, and to 21 subclones derived from one of the lines established from a metastatic focus. Analysis of the 3LL sublines demonstrated a significant antigenic heterogeneity within each tumor population as well as among the different sublines.
AFP‐synthesis induction in mouse hepatomas
Authors: Shipova L. Y. , Gleiberman A. S.
Published: February 2007

Highly differentiated, spontaneous hepatomas occurring in 13‐ to 15‐month‐old CBA mice were unable to produce embryo‐specific serum protein alpha‐fetoprotein (AFP). AFP synthesis, however, was induced in such tumors by minimal mechanical damage (small incisions of the tumors). On the 3rd day following surgery, AFP‐positive cells were detected in two zones: (I) along the inflammatory shaft arising around the peripheral parts of incisions, and (2) near large blood vessels draining the damaged areas. It is suggested that mechanisms regulating AFP synthesis in highly differentiated hepatomas are similar to those of the normal liver and are related to local structural rearrangements.
Short‐term effects of danazol and medroxyprogesterone acetate on cytosol and nuclear estrogen and progestin receptors, 17β‐hydroxysteroid dehydrogenase activity, histopathology, and ultrastructure of human endometrial adenocarcinoma
Authors: Kauppila Anti , Isotalo Hannu , Kivinen Seppo , Stenbäck Frej , Vihko Reijo
Published: February 2007

We measured concentrations of cytosol and nuclear estrogen, as well as progestin receptors and activities of 17β‐hydroxysteroid dehydrogenase (17‐HSD), and examined histopathology and ultrastructure of endometrial carcinoma specimens taken before and after one‐week danazol (200 mg, 3 times daily) or medroxy‐progesterone acetate (MPA, 100 mg daily) treatments in 14 and 16 patients, respectively. A typical progestin effect, a significant increase in the activity of 17‐HSD, was observed after both treatments. The post‐therapy 17‐HSD activities correlated significantly with the pretreatment cytosol progestin receptor concentrations in both treatment groups. Both MPA and danazol decreased the proliferative activity and increased the secretory activity of the malignant epithelial endometrial cells. These biochemical and morphological results support the concept that danazol has progestin‐like actions on the human endometrium, and might therefore be an alternative for hormonal treatment of endometrial carcinoma.
Tissue carcinoembryonic antigen and estrogen receptor in human breast cancer
Authors: Hartfiel Renate , Lopes José Daniel , Lopes Miriam T. P. , Brentani M. Mitzi
Published: February 2007

Sixty‐six human ductal infiltrative breast carcinomas were examined for their estrogen receptor (ER) by the dextran‐coated charcoal procedure and for the presence of carcinoembryonic antigen (CEA) by immunofluorescence with the aid of monoclonal antibodies. Statistically significant differences in CEA positivity were found between stage III (33%) and stage II (19%) tumors (Z = 2.08; p < 0.02) and between ER + (22%) and ER – (37%) tumors (Z = 2.083; p < 0.02). An inverse correlation was observed between positivity and receptor concentrations. No difference in CEA positivity was observed between pre‐ and post‐menopausal women.
An immunohistological method for estimating cell growth fractions in rapid histopathological diagnosis during surgery
Authors: Gerdes Johannes
Published: February 2007

In the past, it has been impossible to carry out immunohistological staining as part of a rapid histopathological diagnosis during surgery because the available methods were too time‐consuming. The present report introduces a rapid indirect immunoperoxidase method that requires only 10–12 min. Thus, the immunological characterization of tumour cells may now be applied as an additional parameter for histopathological diagnosis during surgery. In this first attempt, the monoclonal antibody Ki‐67, which reacts with a human nuclear antigen present in proliferating cells and absent in quiescent cells, was applied with a new immunostaining method and the results were compared with those obtained by the more time‐consuming routine immunohistological method. There was no significant difference between the percentages of Ki‐67‐positive cells determined with the two methods in normal or neoplastic human tissues. Since knowledge of the growth rate of a malignant tumour may be of prognostic and/or therapeutic value in individual cases, it might be useful to routinely apply this new approach for histopathological diagnosis during surgery.
Pregnancy‐associated β1‐macroglobulin: Detection on the surface of ovarian cancer cell lines
Authors: Stimson W. H. , McAdam A. , Lang G. , Dodd J.
Published: February 2007

Pregnancy‐associated β1‐macroglobulin (β1‐PAM) is a serum protein which can be detected at elevated levels in the blood of many ovarian cancer patients. The presence of the antigen was investigated on the surface of 4 human ovarian cancer lines and 32 other human and rodent cell types and lines by means of immunofluorescent staining and anti‐β1‐PAM‐enzyme conjugate. The antigen was detected only on the ovarian lines but no secretion of β1‐PAM into the culture supernatants was observed. The ovarian cancer cells appeared to display the β1‐PAM constantly during culture, and enzymic treatment of the cells indicated that detection of the cell‐bound antigen with antiserum could be, at least partially, eliminated with proteases but not glycosidases or neuraminidase. The results serve to confirm the association of β1‐PAM with ovarian cancer.
Modulatory effects of 5‐azacytidine, phorbol ester, and retinoic acid on the malignant phenotype of human lung cancer cells
Authors: Olsson Lennart , Behnke Olav , Sørensen Hans Rahbek
Published: February 2007

Cloned human cell lines of squamous‐cell lung carcinoma and small‐cell lung carcinoma were treated with 5‐azacytidine (5‐azaC), 12‐0‐tetradecanoyl‐phorbol‐13 acetate (TPA), retinoic acid (RA), or a combination of these drugs, and the effects on cellular morphology, in vitro growth properties, antigenicity, tumorigenicity and metastatic activity in nude mice were studied. Antigenicity was measured by the expression of major histocompatibility antigens (MHC) and of lung‐tumor‐associated antigens.

5‐AzaC treatment resulted in subclones with shorter population doubling times (from 40–50 hr down to 14–20 hr) and increased cloning efficiencies (from < 1% to 5–50%). TPA and RA induced loss of proliferative activity in vitro and tumorigenicity in vivo of both lines. This was morphologically associated with the appearance of an abundance of large vacuolated cells which by DNA‐analysis were all found to be in G0‐G1 phase. However, 2 of the 5‐azaC‐treated subclones were insensitive to both TPA and RA, whereas the remaining subclones (20) all responded like untreated lines to TPA and RA. One of the sublines insensitive to TPA and RA formed metastases in nude mice in contrast to all the other lines used. The density of lung‐tumor‐associated antigens was significantly reduced by TPA‐RA treatment, whereas the expression of MHC antigens was unaffected. In contrast, 5‐azaC resulted in some cases in increased density of MHC antigens. The effects of 5‐azaC on cellular phenotypes were not directly correlated to the total genomic content of 5‐methylcytosine. The data suggest that this experimental system is suitable for studies of phenotypic features of malignant cells as related to cellular differentiation.
Infiltrating mononuclear cells in human breast carcinoma: Predominance of T4+ monocytic cells in the tumor stroma
Authors: Göttlinger Heinrich G. , Rieber Peter , Gokel Josef M. , Lohe Klaus J. , Riethmüller Gert
Published: February 2007

Frozen sections of 52 breast carcinomas were examined for the presence and nature of a leukocytic infiltrate. The number of “common leukocyte (T200‐) antigen”‐bearing cells was remarkably high in the stromal compartment of the carcinomas, whereas the tumor foci themselves were usually infiltrated only sparsely. Approximately 80% of these T200+ stromal cells carried the myelomonocyte lineage‐associated antigens M‐M522 and 63D3 and exhibited “non‐specific esterase” activity. More than 2/3 of the stromal monocytic cells expressed the T4 antigen as verified by monoclonal antibodies (MAbs) directed against different T4‐associated epitopes. A T‐cell analysis showed that T4+ cells clearly outweighed the total number of T cells as defined by anti‐T3 and anti‐TII antibodies. In addition, a correlation became apparent between the HLA‐DR phenotype and the T lymphocyte content of tumor cell areas in the sense that densely infiltrated tumor areas were invariably HLA‐DR+.
Prevalence of human papillomavirus type 16 DNA in cervical carcinoma samples in East Anglia
Authors: Scholl S. M. , Pillers E. M. Kingsley , Robinson R. E. , Farrell P. J.
Published: February 2007

A substantial increase in the incidence of severely dysplastic cervical lesions (CIN 3) has been observed during the period 1975–1982 in the East Anglian region of England. Since patients with severe dysplasia have an enhanced risk of developing cervical carcinoma, it seems possible that a substantial increase in the rate of cervical carcinoma is likely to occur in the near future. Evidence of a relationship between human papillomavirus (HPV) infection and cervical carcinoma has accumulated recently. We have studied the incidence of HPV 16 DNA in cervial tissue samples from patients with cervical carcinoma, severe dysplasias and normal controls. Five out of II invasive squamous carcinomas of the cervix, 3/4 dysplasias and 0/12 normal samples were positive in Southern blot assays for HPV 16 DNA. Some of the tissue samples had as many as 500 copies of HPV 16 DNA per cell. The amount of HPV 16 DNA present correlated with the aggressiveness of tumour growth.
Changes in fibronectin synthesis and binding distribution in SV40‐transformed human keratinocytes
Authors: Edelman B. , Steinberg M. L. , Defendi V.
Published: February 2007

We have studied the synthesis and distribution of fibronectin in human epidermal keratinocytes infected by SV40, a system in which the acquisition of transformed properties occurs in a sequential and progressive manner. Immunofluorescence studies showed that cultured unifected keratinocytes do not exhibit fibronectin on the superficial cell surface, but that virus‐infected cells come to display superficial fibronectin‐containing cables in a density‐dependent manner after a certain point in the transformation process. In contrast, organized arrays of fibronectin‐containing fibrils associated with the cell‐substrate attachment complex were seen in uninfected keratinocytes and in virus‐infected cells at all stages of the transformation process. Studies of fibronectin synthesis using metabolic labelling of cell proteins with 35S methionine showed that viral infection caused a striking increase in overall fibronectin synthesis, although with a much higher proportion of newly synthesized fibronectin being secreted into the cell culture medium than in the case of the uninfected cells.
Expression of ki‐ras oncogene in tumor cell variants exhibiting different metastatic capabilities
Authors: Kris R. M. , Avivi A. , Bar‐Eli M. , Alon Y. , Carmi P. , Schlessinger J. , Raz A.
Published: February 2007

The expression of oncogenes in well‐characterized 616 melanoma and UV‐2237 fibrosarcoma cell variants that exhibit distinct metastatic properties was explored. The search for the expression of II different oncogenes revealed that the major oncogene in those two tumor systems is the Kirsten‐ras (ki‐ras). The results indicate that the amounts of specific ki‐ras mRNA and the p21 protein are similar in both low‐ and highmetastatic counterparts. These results suggest that in these systems there is no apparent direct correlation between the amount and expression of the major cellular oncogene so far identified and the metastatic potential of these tumor cells.
Identification of Marek's disease virus‐specific antigens in Marek's disease lymphoblastoid cell lines using monoclonal antibody against virus‐specific phosphorylated polypeptides
Authors: Ikuta Kazuyoshi , Nakajima Kazuhiro , Naito Matao , Ann Soo H. , Ueda Shigeharu , Kato Shiro , Hirai Kanji
Published: February 2007

For identification of the antigens specific to Marek's disease virus (MDV) in virus‐non‐producer lymphoblastoid cell lines established from a tumor of Marek's disease (MD), hybridomas producing monoclonal antibodies (MABs) against the antigens were isolated. Immunogens for preparation of the hybridomas were purified from the lysate of an MD‐lymphoblastoid cell line, MSB I, by affinity chromatography coupled with chicken anti‐MDV serum immunoglobulin G. Three of the MAbs obtained, MB1, MB2 and MB3, were specific to MDV by immunofluorexence test. An immunofluorexence test using MB2 antibody showed that immunofluorexence‐positive cells in non‐producer MD‐lymphoblastoid line cells became detectable when the culture temperature was shifted from 41° C to 33° C or when treatment with 5‐iodo‐2‐deoxyuridine (IUdR) was performed, indicating that the antigen reactive with MB2 antibody is an MDV‐specific early antigen. This temperature shift of IUdR‐treatment did not induce other MDV‐specific antigens, such as late gene products of MDV, detected with MAbs. MB1 and MB2 antibodies immunoprecipitated 4 MDV‐specific phosphorylated polypeptides with molecular weights (MWs) of 43,000 (43kd), 39kd, 36kd and 24kd from chick embryo fibroblasts productively infected with virulent MDV. In the place of 43kd, phosphorylated 44kd polypeptide was precipitated from avirulent MDV‐infected fibroblasts. However, MB3 antibody did not precipitate any MDV‐specific polypeptides from infected fibroblasts. These results suggest that the phosphorylated polypeptides are MDV‐specific polypeptides predominantly expressed in non‐producer MD‐lymphoblastoid cell lines.
Studies on clonal heterogeneity in two spontaneously metastasizing mammary carcinomas of recent origin
Authors: Brodt P. , Parhar R. , Sankar P. , Lala P. K.
Published: February 2007

We have studied the clonal heterogeneity of 2 spontaneously metastasizing mammary carcinomas which recently arose spontaneously in C3H/He female retired breeders. Cells of early (2nd to 5th) transplant generations of these tumors were cloned by a combination of semi‐solid agarose colony formation and limiting dilution techniques.

Growth characteristics of the various clones in vitro and their tumorigenicity in vivo were evaluated. Subsequently, the role of host immunity and of interclonal interactions in regulating growth of the different clones in vivo were analyzed. We found that, whereas all 16 clones isolated from one tumor (T‐58) grew rapidly in vivo and in vitro, 10 clones isolated from the second tumor (MT‐2) showed a wide disparity in their growth rates in vivo. Taken together, these clones could generally be divided into 3 categories: (I) rapidly growing lines which grew in vivo at rates similar to or higher than those of the parental line; (2) slow‐growing lines which grew more slowly than the parental line; and (3) non‐growers which failed to produce tumors in vivo with doses of up to 5 × 106 cells injected either s. c. or i. v. but grew in vitro at rates comparable to the parental line. No correlation could be established between the various growth potentials exhibited by these tumor lines and tumor cell morphology in vitro and in vivo, as determined by light and electron microscopy. Sublethal irradiation (550‐650 R) of young animals prior to tumor inoculation, or before inoculation of tumor cells into old, low NK syngeneic mice, failed to modify the growth of slow or non‐growing lines in vivo, indicating that host cellular defense mechanisms against the clones, if existent, were not mediated by NK or radiosensitive B or T cells. When clonal interactions were studied by the simultaneous injection of different clones in vivo at different s. c. sites, we found that a slow‐growing line failed to modify the growth rate of a rapidly growing line, but accelerated the growth of a second slowgrowing line injected simultaneously on the contralateral side, and that this enhancement of tumor growth was radioresistant. A mixture of these 2 lines also grew more rapidly than the individual lines alone. Our findings suggest that phenotypic variations in tumorigenicity can be found in clonal lines derived from spontaneous primary tumors and that these variations are not related to cell cycle properties as measured in vitro. Furthermore, it appears that growth properties of these tumor lines in vivo are not easily modifiable by manipulation of the host immune reactivity but may be subject to interclonal growth regulation.
Neoplastic transformation of human diploid fibroblasts (KMST‐6) by treatment with 60Co gamma rays
Authors: Namba Masayoshi , Nishitani Koji , Hyodoh Fuminori , Fukushima Fujiko , Kimoto Tetsuo
Published: February 2007

Normal fibroblasts (KMS‐6) derived from a human embryo were transformed in culture into neoplastic cells (KMST‐6) by repeated treatment with 60Co gamma ray irradiation. Repeated treatment was necessary to obtain transformation. Control normal cells exhibited normal karyotype (46, XX) and stopped dividing due to cellular ageing at the 40th passage. The transformed cells are presently growing indefinitely (140th passage) and exhibit prominent karyologic aberrations, both numerical and structural. These 2 characteristics, indefinite growth and abnormal karyotype, are thought to be the most important parameters for neoplastic transformation of human fibroblasts. Other indispensable parameters are the presence of active mitotic figures on confluent cell sheets and colony‐type morphology. Transformed cells grow into colonies with relatively smooth edges, while normal fibroblasts form colonies with jagged edges, due to the protrusion of growing fibroblasts. Other parameters, such as elevated plating efficiency, enhanced colony formation in soft agar, low serum requirement for growth, high saturation density, and acquisition of transplantability, are not reliable in the early stages of transformation. These parameters probably appear at rather later stages of transformation following several cell divisions. Among other characteristics, the transformed KMST‐6 cells exhibit a B‐type isozyme pattern of glucose‐6‐phosphate dehydrogenase, lactate‐dehydrogenase isozyme pattern of human origin, no evidence of viral infection and no production of C‐type virus particles.
Behaviour of dendritic reticulum cells possessing immunoreactive acid cysteine‐proteinase inhibitor in human lymphoid secondary follicles and in follicular‐centre cell lymphomas
Authors: Alavaikko M. , Aine R. , Rinne A. , Järvinen M. , Blanco G. , Apaja‐Sarkkinen M. , Hopsu‐Havu V. K.
Published: February 2007

In our previous studies acid cysteine‐proteinase inhibitor (ACPI) was shown to be a common characteristic of human squamous epithelia and dendritic reticulum cells (DRC) in lymphoid secondary follicles. In the present study, the behaviour of ACPI‐immunoreactive DRC in reactive lymphoid secondary follicles and neoplastic follicles of follicular‐centre cell (FCC) lymphomas was compared by the peroxidase‐antiperoxidase method. The secondary follicles in reactive lymphoid tissues revealed a staining pattern characteristic of DRC comparable with the results of other studies. Contrary to this, the number of ACPI‐positive DRC in the neoplastic lymphoid follicles was greatly reduced. This was occasionally accompanied by a weak reaction and in many cases the DRC were totally abolished. Moreover, morphological aberrations were observed in ACPI‐positive DRC, especially diminution and shortening of dendritic processes. On occasion, ACPI‐positive cell types were observed, which may represent intermediate forms between DRC and fibroblastic reticulum cells. Only one case‐with a rather favourable clinical outcome (out of 41 follicular FCC lymphomas) exhibited an ACPI‐positive DRC pattern comparable with that of reactive lymphoid follicles. Thus it seems that the ACPI‐immunohistochemistry of DRC provides an additional tool for discerning the difference between reactive and neoplastic lymphoid follicles. This method is of particular value since it can be used with formalin‐fixed, paraffinembedded tissues. There was a tendency among the follicular FCC lymphoma patients towards a better survival rate for those with ACPI‐positive DRC than for those lacking this cell type, although no statistically significant differences emerged from this limited material.
Generation of Epstein‐Barr virus antigen‐specific suppressor T cells in vitro
Authors: Sundar Syam K. , Menezes José
Published: February 2007

Immunosuppression is a commonly observed phenomenon in Epstein‐Barr virus (EBV)‐associated disorders and malignancies. The purpose of this study was to determine whether EBV antigens could generate suppressor cell activity in vitro. Pepripheral blood lymphocytes (PBL) were first treated with various concentrations of EBV antigens or culture medium for 5 days and then with mitomycin C. The cells were then washed and tested for their ability to abrogate the blastogenic response of fresh, autologous PBL to previously determined optimal concentrations of EBV antigens. It was found that excess of both EBV antigens tested (soluble antigen and virus particles) induced suppressor cells, while optimal antigen concentrations failed to do so. In addition, PBL incubated with excess of EBV antigens for 10 days, without mitomycin treatment, inhibited the response of fresh autologous lymphocytes to EBV antigens. The generated suppressor cells were found to be antigen‐specific since they inhibited the response of sensitized lymphocytes to the inducing antigen only. Moreover, experiments performed using purified lymphocyte subpopulations indicated that the suppressor activity was associated with T‐cell populations. Using T‐cell specific monoclonal antibodies, we further determined that the inhibitory activity was due to suppressor (OKT 8+) T‐lymphocytes; treatment of T‐lymphocyte populations (exhibiting suppressor activity) with OKT 8 antibody and complement abrogated the inhibitory effect of these populations on the response of sensitized lymphocytes to EBV antigens. Taken together, these observations suggest that similar supressor cells may be at least partly responsible for the immunosuppression observed in patients with an antigenic overload, particularly during persistently active virus infection or malignancy.
Alterations in the phosphoprotein composition of tumor cell clones induced by cell culture techniques used routinely in assessing metastatic behavior in vivo
Authors: Greig Russell G. , Caltabiano Lyn , Feild John , Reid Robert , Poste George
Published: February 2007

To investigate whether the cell dispersion techniques commonly employed to harvest monolayer cultures of tumor cells for injection into experimental animals might induce alterations in cellular biochemistry, we compared the phosphoprotein profiles of 6 B16 melanoma clones of distinct metastatic potential using 2‐D gel electrophoresis after growth in monolayer culture, after suspension by treatment with trypsin/EDTA and after injection of suspended cells into syngeneic mouse plasma. Trypsin/EDTA treatment and subsequent exposure to syngeneic mouse plasma induced significant alterations in phosphoprotein composition in all clones. Most alterations were quantitative, involving either enhanced or diminished expression of specific phosphoproteins, but qualitative changes involving expression of novel phosphoproteins were also observed. None of the changes in phosphoprotein composition correlated with metastatic potential. The principle alteration induced in all clones by trypsin/EDTA involved enhanced phosphorylation of an NP‐40‐soluble component with a molecular weight of 79,000 and an isoelectric point of 6.3 [pp 79 (6.3)]. This determinant was detected in extracts of BI6 monolayer cultures but its level of phosphorylation was enhanced significantly by trypsin/EDTA treatment and by exposure of the harvested cells to syngeneic mouse plasma. These data indicate that procedures commonly employed to harvest tumor cells for assay of tumorigenic and metastatic potential may provoke extensive alterations in phosphoprotein composition and that biochemical investigations of tumor cells grown in monolayer culture may not accurately reflect the metabolic status of the same cells immediately prior to and following i.v. injection into experimental animals.
Characterization of tumor progression from threshold tumor inocula: Evidence for natural resistance
Authors: Brown Garth W. , Chow Donna A.
Published: February 2007

An examination of the variant generation and selection hypothesis of tumor progression was undertaken using the NK‐sensitive, NAb‐sensitive SL2‐5 lymphoma in the threshold inoculum model of tumor progression. Tumor cells obtained from the i.p. injection site expressed increased heterogeneity for sensitivity to syngeneic NAb and to NR measured in the 131 IUdR‐labelled tumor elimination assay. Cells retrieved from the s.c. injection site exhibited reductions in sensitivity to NR which correlated with decreases in sensitivity to syngeneic NAb and NK cells in vitro. These data confirm and extend our previousobservations with the NK‐resistant L5178Y‐F9 lymphoma and further substantiate the evidence for the participation of NAb and NK cells in host‐mediated anti‐tumor resistance. Characterization of the model revealed that the selection for reduced sensitivity to NR in thymus‐depleted AT × BM mice and normal animals could not be distinguished, suggesting that thymus‐independent mechanisms may be major contributors to the surveillance of nascent tumors. The decreases in susceptibility to NR occurred in a stepwise and time‐dependent manner in accord with the sequential multistage concept of progression. Furthermore, the selection for tumor cells which exhibited reductions in sensitivity to NR correlated with selection for increased tumorigenicity, in keeping with the idea that progression is associated with development towards an increasingly autonomous tumor.
Effects of diazoxide‐induced reversible diabetes on chemically induced autochthonous mammary carcinomas in sprague‐dawley rats
Authors: Berger M. R. , Fink M. , Feichter G. E. , Janetschek P.
Published: February 2007

The effect of oral administration of diazoxide on rats bearing mammary carcinomas induced by dimethylbenzanthracene (7,12‐DMBA) or methylnitro sourea (MNU) was investigated. Administration of 300 mg/kg diazoxide caused mild reversible diabetes with maximum glucose levels of 305 ± 74 (control: 119 ± 12) mg/dl and related insulin levels of 15 ± 5 (control: 24 ± II) μ U/ml after 4 hr in tumor‐bearing animals. Following the same dose of diazoxide a more than 90% inhibition of tumor growth was observed in 7, 12‐DMBA‐ and MNU‐induced autochthonous rat mammary carcinomas as well as remission of the median total tumor volume per group in 7, 12‐DMBA‐induced lesions. Frequency, onset of remissions and median remission duration proved to be dose‐dependent in 7,12‐DMBA‐induced mammary carcinoma and, with the exception of the median remission duration, in MNU‐induced tumors too. After cessation of diazoxide application, 30% rebound responses were observed in 7, 12‐DMBA‐induced tumors of animals that had had a first remission due to diazoxide. Application of insulin (2 IU per rat) together with diazoxide (300 mg/kg) reversed the tumor‐inhibiting effect of diazoxide in MNU‐induced tumors. The diazoxide effect might in part be due to a decrease in the percentage of proliferating cells caused by insulin depletion as indicated by a lower amount of cells in S‐phase, as measured by DNA‐flow cytometry. Marked toxicity was observed after effective doses of diazoxide; the experiments indicate that induction of reversible diabetes might be a useful tool in the treatment of hormone‐dependent mammary carcinoma.