The establishment of lymphoblastoid lines from adult and fetal human lymphoid tissue and its dependence on EBV

Continuous human lymphoblastoid cell lines (LL), derived from lymphoid tissue or peripheral blood of 20 adults without histories of recent infectious mononucleosis at a frequency close to 100 %, were examined for the presence of Epstein-Barr virus (EBV) using immunofluorescence methods for the detection of EBV-dependent cell membrane and EB viral capsid antigens. All lines except one were found to be EBV carriers in the initial tests, but the antigens gradually disappeared in most during the course of an observation period of 4 months. Only eight lines maintained the initial percentage of antigen-containing cells. The results of the two immunofluorescence tests, performed simultaneously on each cell pool, were concordant in all instances. Sera were available from 16/20 donors. All cell donors, except one, possessed antibodies to EBV as an indication of prior EBV infections. The growth-promoting role of EBV in the establishment of LL was supported by studies with fetal lymphoid tissue cultured by the same grid method which yielded the high frequency of LL from adults. In sharp contrast to the results obtained with adult lymphoid tissue, no lines were established from 20 fetuses aged 13-20 weeks. However, when such tissue was exposed to a cell-free filtrate prepared from an EBV-carrying LL lymphoblastoid cell, lines were established in some instances. Filtrate from an EBV-negative line inoculated into parallel cultures failed to promote the establishment of LL. The results indicate that EBV infection in vivo or in vitro may be a prerequisite for the indefinite growth of lymphoblastoid cells in vitro and that EBV infections, as a rule, are not vertically transmitted.     

Évolution in vitro et cancérisation des cellules pulmonaires de souris

Evolution in vitro and malignant transformation of mouse lung cells In nine independent experiments, performed during the last five years, mouse lung tissue from adult C57Bl/6/J females was explanted in vitro in order to establish long-term cultures. The cultures were started with tissue fragments embedded in chick plasma clot and were continued as monolayer cultures, treated during passages either by tryp-sinization or mechanical dispersion. Media prepared with the 199 or NCTC 109 synthetic mixtures, completed with horse or calf serum, were used routinely. In all attempts undertaken, we obtained permanent cell lines which were: P1, P3, P5 and P7 trypsinized, P2, P4, P6, P8 non-trypsinized and P4bis trypsinized only once at the start. After 3 to 4 months of culture in vitro, cells having abnormal karyotypes, with chromosome numbers spread over the 45–100 area (and higher), accumulated progressively replacing the normal cells. The wide dispersion of chromosome numbers persisted many months and was evident in all the lines when checked between the 27th and 32nd months of culture. Biarmed, marker chromosomes were consistently found in some of the lines. Assays for malignancy were undertaken with all lines between the 7th and 12th months of culture, by subcutaneous inoculation of 5 × 10⁶ cells in adults and 1 × 10⁶ cells in newborn mice. All lines, except P6 and P7, proved to be capable of producing transplantable, sarcomatous tumors. P7 gave finally tumors after 16 months in vitro. P6 remained non-malignant until the end of the third year in vitro in spite of its highly abnormal karyotype. As a rule, chromosomal modifications preceded the appearance of malignancy. Morphologically, P cells evolved during the long-term culture toward more homogeneity. However, characteristic differences of cellular aspect persisted between different lines as well as inside a single line, as was the case for P4bis which maintained permanently a composite, double morphology. The P lines differed also consistently in their malignancy as evaluated by the speed of tumor growth and by the minimum tumor producing cell number. A relation existed apparently between malignancy and cell morphology: cells of highly malignant lines had in vitro a pronounced spindle-like aspect and easily produced multilayer, intermingled growth, whereas the non-malignant P6 line maintained monolayer, pavementlike structures even in older cultures. The differences between P lines were reflected also in the histological aspect of the tumors: for example, giant cells were proper to the P4bis line in vivo as well as in vitro; massive production of collagen was proper to the P3 and P4 lines. Some of the P lines revealed C type virus particles. The possible role of viruses, especially of endogenous origin, in the “spontaneous” transformation of long-term mouse cell cultures is considered and discussed.     

Nasopharyngeal carcinoma (NPC). I. Types of cultures derived from tumour biopsies and non-tumorous tissues of Chinese patients with special reference to lymphoblastoid transformation

Biopsy specimens of nasopharyngeal carcinoma (NPC) from Chinese patients were sent from Hong Kong to Lyons and cultured in vitro. Four different types of culture were obtained: 1) epithelial growth of short duration (4 to 12 weeks) and limited extent around the explants; 2) early lymphocytic production (ELP) which lasted for 1 to 3 weeks; 3) fibroblastic cultures (either as primary or secondary to the epithelial growth); 4) “lymphoblastoid transformation” which occurred in 28% of the cultures and resulted in the establishment of long-term free-floating cell-lines. A few of these lines were intermittently dependent on the presence of fibroblasts during crises, while most of them were entirely independent. Biopsy specimens from head and neck tumours other than NPC, from hypertrophied and inflamed tonsils removed from children, and from apparently tumour-free nasopharyngeal mucosa gave rise to a similar spectrum of cultures, including the establishment of long-term lymphoblastoid cultures. The various lines thus obtained exhibited the presence of a herpes-type virus (HTV). It is proposed that the establishment of permanent lymphoblastoid lines is caused by the presence, in the original material, of an HTV having “transforming” properties. The significance of these results and the nature of the serological association between NPC and HTV are discussed.     

Sensitivity of Epstein-Barr virus (EBV) producer and non-producer human lymphoblastoid cell lines to superinfection with EB-virus

Twenty-nine lymphoblastoid lines and one IgE-producing myeloma line of human origin were exposed to Epstein-Barr virus (EBV) concentrates in vitro. The adsorption of the virus to the outer cell membrane was assessed by counting the number of direct membrane fluorescence-positive cells immediately after infection and by the direct radioimmune membrane labelling method. Reference reagents were derived for both tests from the same serum (“Agnes”), containing antibodies against EB-viral envelope and capsid antigens. The intracellular course of infection was followed by counting the number of cells that responded with the development of early antigen (EA) 48 h after infection. The 11 lymphoblastoid lines that produced no EBV-determined membrane and early antigens adsorbed the virus, although there were quantitative differences between them. EA-positive cells appeared in significant numbers in only seven of them, however. Four lines remained EA negative in spite of a relatively good adsorbing capacity. The IgE-producing myeloma line showed neither virus adsorption nor EA development. Eighteen lymphoblastoid lines were “producers”, or “abortive producers”, i.e.a small proportion of the cells continuously generated two or three of the immunofluorescence-detectable viral antigens, MA, EA and VCA. Nine lines failed to adsorb significant virus quantities and showed no certain increase of EA-positive cells. The resistance of these lines to superinfection is probably determined at the level of viral receptors. Five lines showed a relatively good virus adsorption, but this was not followed by any significant increase in the number of EA-positive cells. Four lines showed good adsorption and also responded with a significant increase in the number of EA-positive cells. The same responses can thus be found to EBV-superinfection in producer and non-producer lines, but the producer lines show a strong preponderance of superinfection-resistant lines with an adsorption block at the receptor level.     

Transformation of tracheal epithelium exposed in vitro to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)

Using an organ culture/cell culture system, we transformed rat tracheal epithelial cells in vitro by exposure to MNNG. Ten tracheal organ cultures per group were exposed twice (at days 3 and 6) to 0,0.001, 1.0 or 10.0 μg MNNG/ml of medium. Following this exposure, the explants were placed on the bottom of culture dishes to initiate epithelial cell outgrowths and establish primary cultures. Each explant was replanted 8–10 times to produce multiple outgrowths. The number of primary cultures and the number of subsequently established cell lines obtained was carcinogen-dose-dependent. The average number of primary epithelial cell cultures per explant after exposure to 0, 0.001, 1.0 and 10.0 μg MNNG/ml was 1.3, 1.5, 3.3, and 4.6, respectively. The average yield of cell lines per explant in these groups was 0, 0.8, 1.3, and 2.0, respectively. It is apparent that cell lines could only be established from carcinogen-exposed epithelium. These cell lines are currently being tested for tumorigenicity in vivo. To date, of 35 cell lines tested between the 5th and 15th passages, 5 cell lines from the 10 μg MNNG/ml group and 2 from the 1.0 μg MNNG/ml group have produced palpable tumors upon injection into immuno-suppressed recipients.     

Tumorigenicity of human lymphoblastoid cell lines,acquired during in vitro culture and associated with chromosome gains

Tumorigenicity of human lymphoma and lymphoblastoid B-cell lines was assessed by their ability to form growing and transplantable masses on subcutaneous inoculation into neonatally thymectomized, Ara-C-protected, totalbody-irradiated mice. By these criteria, 12 lines of known malignant origin were tumorigenic, 11 lymphoblastoid lines, tested after less than one year of in vitro growth, were non-tumorigenic and 8/18 long-established lymphoblastoid lines produced transplantable tumours. All of the long-established lines had acquired karyotypic changes on prolonged culture, the predominant characteristic being a gain of whole chromosomes or of major chromosome segments. None showed the classical 8:14 translocation associated with Burkitt's lymphoma. Comparisons with nontumorigenic precursors (recovered from liquid nitrogen storage) and with other non-tumorigenic but chromosomally abnormal, lymphoblastoid lines suggest that imbalance of the dosage of genes carried on chromosomes 7,8 and 9 may be important in determining the tumorigenic phenotype.     

Establishment of six new human biliary tract carcinoma cell lines and identification of MAGEH1 as a candidate biomarker for predicting the efficacy of gemcitabine treatment

The aim of this study was to establish new biliary tract carcinoma (BTC) cell lines and identify predictive biomarkers for the potential effectiveness of gemcitabine therapy. Surgical specimens of BTC were transplanted directly into immunodeficient mice to establish xenografts, then subjected to in vitro cell culture. The gemcitabine sensitivity of each cell line was determined and compared with the genome‐wide gene expression profile. A new predictive biomarker candidate was validated using an additional cohort of gemcitabine‐treated BTC cases. From 55 BTC cases, we established 19 xenografts and six new cell lines. Based on their gemcitabine sensitivity, 10 BTC cell lines (including six new and four publicly available ones) were clearly categorized into two groups, and MAGEH1 mRNA expression in the tumor cells showed a significant negative correlation with their sensitivity to gemcitabine. Immunohistochemically, MAGEH1 protein was detected in three (50%) out of six sensitive cell lines, and four (100%) out of four resistant cell lines. In the validation cohort of gemcitabine‐treated recurrence cases, patients were categorized into “effective” and “non‐effective” groups according to the RECIST guidelines for assessment of chemotherapeutic effects. MAGEH1 protein expression was detected in two (40%) out of five “effective” cases and all four (100%) “non‐effective” cases. We have established a new BTC bioresource that covers a wide range of biological features, including drug sensitivity, and is linked with clinical information. Negative expression of MAGEH1 protein serves as a potential predictive marker for the effectiveness of gemcitabine therapy in BTC. (Cancer Sci 2010; 101: 882–888)     

Intrinsic gene changes determine the successful establishment of stable renal cancer cell lines from tumor tissue

Human tumor cell lines, especially those with complete data and follow‐up, are important tools in tumor biological studies. Clear cell renal cell cancer (ccRCC) is not sensitive to radiotherapy or chemotherapy, and treatment of patients with distant metastasis relies on targeted therapy. Here, we report the establishment of seven new ccRCC stable cell lines that were continuously cultured for more than 20 generations among 81 cases of renal cell cancer. Moreover, gene expression and methylation in the established cell lines, in those that had a finite in vitro life span of less than 10 generations, and in cells that originated from the same culture at different generations were profiled using microarrays. Genes including SLC34A2, SEPP1, SULT1C4 and others were differentially expressed in established cell lines and finite cell lines, and changes in their expression might be caused by methylation or demethylation. The expression level of SLC34A2 was related not only to the life span in vitro culture but also to tumor size. Additionally, six of the seven new ccRCC cell lines had VHL deletions or termination mutations. So in addition to the establishment of seven new ccRCC cell lines with complete clinical data, we conclude that genes such as SLC34A2 and VHL play key roles in the continuous in vitro growth and development of ccRCC.     

Transplantable mouse tumor line induced by injection of SV40-transformed mouse kidney cells

A transplantable tumor of inbred mice was obtained by inoculating BALB/c mice subcutaneously with SV40-transformed mouse kidney (mKS-A) cells. Tumors were produced by mKS-A cells in the 71st cell culture passage, but not by cells in the 26th passage. The tumor line has been serially passed in BALB/c mice 14 times. In vitro cell culture lines were derived from tumors after 1, 2, 8, 10 and 12 passages in mice. The tumors, as well as the In vitro tumor cell lines, contained SV40 T-antigen, and sera from the tumor-bearing mice contained antibodies to the SV40 T-antigen. SV40 was rescued from the In vitro tumor cell lines after fusion with green monkey kidney (CV-1) cells in the presence of UV-irradiated Sendai virus. The In vitro tumor cell lines derived from mouse passages 8, 10 and 12 were used as SV40 virus; 2) SV40-transformed cell lines; 3) primary mouse (BALB/c or Yale Swiss) kidney cells, or 4) primary mouse (BALB/c or Yale Swiss) embryo cells. These results showed that the tumor line and the In vitro tumor cell lines have the transplantation antigen.     

Production and characterization of marmoset lymphoblastoid interferon

We describe the production of marmoset lymphoblastoid cell interferon (IFN). Optimal yields of IFN were obtained when EBV-transformed lymphoblastoid cell lines (LCL) at a cell density of 1 × 10⁶/ml were incubated with 100 HAU Sendai virus/ml for 24 h. Sendai-virus-induced marmoset lymphoblastoid cell IFN was acid-stable and exerted antiviral activity on both homologous and heterologous human cells, thus allowing its classification as IFN α. Marmoset lymphoblastoid cell IFN did not inhibit the growth of herpesvirus-transformed marmoset T- or B-cell lines, but markedly enhanced marmoset NK-cell activity against human myeloid leukemia target cells. The use of lymphoblastoid cell IFN in marmosets in vivo may contribute to an understanding of the pathogenic role of IFN in herpesvirus-induced lymphoproliferative diseases.     

Comparative cytogenetic studies of normal and leukemic lymphoblastoid cell lines during the course of their establishment.

A comparative chromosomal analysis was made of 10 human lymphoblastoid cell lines, four of which originated from normal donor lymphocytes and six of which were from leukemic peripheral blood. For comparison of lymphoblastoid cells with respect to their normal or leukemic origin, cytogenetic studies have been carried out regularly since the beginning of the culture during more than 3 years. Samples were drawn during the three phases previously described for the establishment of these lines. The chromosome distribution remained diploid for at least 2 years in normal cell lines, and the cells were euploid. In contrast, an important variability of the chromosome set was demonstrated during the same period in leukemic cell lines. Moreover, in these lines, it was always possible to observe a nonsystemic pseudodiploidy. After 2 years, a clonal evolution was described in both types of cell lines that carried at least one marker. With a controlled-heating denaturation technique, it was possible to identify the markers as specific to each cell line. The cells with marker chromosomes appeared to have a selective advantage of growth.     

Classification and biological nature of established human hematopoietic cell lines.

Over 200 established human hematopoietic cell lines of normal and malignant origin have been investigated by morphological and functional parameters. Employing morphology as the overriding parameter four types of lines were identified. (1) Lymphoblastoid cell lines, derived from normal and neoplastic hematopoietic tissue, were characterized by the wide morphologic flexibility of individual lymphoblastoid cells, constant association with Epstein-Barr virus (EBV), polyclonal derivation, differentiation for immunoglobulin production (secretion) and their diploids. (2) Lymphoma cell lines. This type of line was established at a high frequency from Burkitt's lymphoma and rarely from other types of lymphoma, but never from patients without malignancy or with non-lymphoma malignancies. Important characteristics were morphologic stereotypia within each line, monoclonal derivation, common but not obligatory association with EBV, variability in the expression of Ig synthesis (no production, or membrane bound Ig, or secretion) and aneuploidy. (3) Myeloma cell lines could only rarely be obtained from patients with myeloma. The basis for classification of these lines is their production of Ig identical to the myeloma protein in vitro. Other important distinguishing features were: plasma cell morphology, absence of EBV and aneuploidy. (4) The leukemia cell line (MOLT 4) was the only line with T-cell characteristics and was easily distinguished from the other types. Important characteristics were a typical surface ultrastructure, absence of EBV and absence of immunoglobulin production, Individual lymphoblastoid lines were in principle identical whereas each line of the other three types had its own characteristic profile. The phenotypic characteristics of the lymphoblastoid lines were very stable during prolonged serial cultivation. Only in a few cases were secondary chromosomal, functional or morphologic alterations noted. We conclude that EBV-carrying lymphoblastoid lines can be obtained from non-neoplastic precursor cells from healthy as well as from diseased individuals. Lymphoma, myeloma and leukemia lines are only obtained from the respective neoplastic tissue but generally at a low frequency. With the exception of Burkitt's lymphoma, malignant hematopoietic tissue and leukemia frequently give rise to established cell lines in vitro of the lymphoblastoid type rather than lines derived from the neoplastic cells;     

Biologically relevant phenotypic changes and enhanced growth properties induced in B lymphocytes by an EBV strain derived from a histologically aggressive Hodgkin's disease

Epstein‐Barr virus (EBV) isolates show a wide genomic heterogeneity, and a key issue is whether distinct strain variations may contribute to the development and/or malignancy of EBV‐related disorders. Herein, we report on the virologic and biologic characterization of an EBV strain derived from a cyto‐histologically aggressive EBV‐related Hodgkin's disease (HD) (case HD‐3) showing a high number of “anaplastic” Reed‐Sternberg cells expressing markedly high levels of CD30, CD40 and LMP‐1. The HD‐3‐derived EBV showed strong in vitro immortalizing properties, as suggested by the unusually high number of spontaneous lymphoblastoid cell lines (LCLs) obtained from the patient. Immunofluorescence and immuno‐cytochemical analyses showed that HD‐3 LCLs expressed significantly higher levels of CD23, CD30, CD38, CD39, CD40 and CD71 antigens and CD54 and CD58 adhesion molecules than B95.8 LCLs. In contrast, the expression of CD11a, CD24, CD95, bcl‐2, LMP‐1 and EBNA‐2 was similar in both groups of LCLs. These phenotypic changes are consistent with the induction of a pronounced activation status and are not dependent on the cellular background, having been closely reproduced by the same virus in LCLs from an unrelated donor (DEN‐HD‐3 LCLs). HD‐3 LCLs were able to grow in vitro at low serum concentrations (up to 0.1%) and were significantly more clonogenic in soft agarose than B95.8 LCLs. Moreover, although no evidence of tumor formation was observed in nude mice injected with B95.8 LCLs, all 5 spontaneous LCLs of patient HD‐3 and the 2 DEN‐HD‐3 LCLs grew in transplanted animals as lymphoproliferations composed of EBER⁺, LMP‐1⁺ cells. Our findings indicate that the biologic properties of the HD‐3 EBV strain are significantly different from those of the B95.8 virus and may have contributed to the cytologic and histo‐pathologic malignancy of this HD case. Moreover, molecular characterization of the HD‐3 EBV genome identified a 63‐bp deletion within the 3′ end of the LMP‐1 gene as a likely significant change that may be responsible, at least in part, for the biologically relevant phenotypic modifications and enhanced in vitro and in vivo growth potential induced in B lymphocytes by this virus strain. Int. J. Cancer80:240–249, 1999. © 1999 Wiley‐Liss, Inc.     

Mitogenic effect of the 15-kDa gross cystic disease fluid protein (GCDFP-15) on breast-cancer cell lines and on immortal mammary cells

The biological significance of a major protein component in the fluid of gross cystic breast disease and a recognized marker of apocrine metaplasia, i.e. the 15-kDa glycoprotein (GCDFP-15), is presently unknown. We have added GCDFP-15 to cell culture medium and tested its effect on proliferation of 4 human breast-cancer cell lines (MCF7, BT474, MDA-MB231 and T47D) and a “normal” human immortal breast-cell line (MCF10A). These breast-cell lines showed a mitogenic response to GCDFP-15 (10 μ/ml). GCDFP-15 enhanced cell growth of the MCF10A, MCF7, BT474 and MDA-MB231 cell lines at both 48 and 96 hr of exposure. The glycoprotein exerted a mitogenic effect on the T47D cell line at 48 hr but not at 96 hr. This may be due to an auto-regulatory effect of endogenous GCDFP-15 synthesized by the T47D cells. GCDFP-15 was ineffective on 2 colon-cancer cell lines (HT29 and NIC-H7I6), on the IMR32 neuroblastoma cell line and on the NIC-H209 small-cell lung carcinoma cells. A separate major breast cystic disease fluid protein of 24 kDa (GCDFP-24) was tested, following the same experimental design, on the 5 breast-cell lines, and showed no mitogenic activity. The mitogenic effect of GCDFP-15 observed in this study in both “normal” and malignant breast epithelial cells suggests a possible relationship between apocrine metaplasia in breast cystic disease and the development of breast epithelial hyperplasia. In addition, a possible role of GCDFP-15 in breast-cancer progression should be considered. © 1995 Wiley-Liss, Inc.     

Tumor metastases and cell-mediated immunity in a model system in DBA/2 mice. I. Tumor invasiveness in vitro and metastasis formation in vivo

A syngeneic model system for the study of tumor metastases and cell-mediated immunity is described. The system consists of two related, chemically induced murine lymphomas, the non-metastasizing parental line Eb and its metastasizing variant ESb. An unrelated, chemically induced tumor (MDAY) is included for specificity controls. Serological typing revealed that both Eb and ESb were of T lymphoid origin and expressed the H-2K and H-2D molecules of the host strain DBA/2. By various electron microscope techniques, morphological differences were observed between the two cell lines. In comparison to Eb cells, ESb tumor cells had a more polymorphic nucleus with many in vagi-nations of the nuclear envelope and a more prominent expression of microvilli on the cell surface. An in vitro organ culture test for tumor invasiveness, presented here for the first time in a syngeneic murine system, revealed that ESb but not Eb tumor cells had the ability to attach to and invade normal tissue. Accordingly, ESb tumor cells showed higher malignancy in vivo. This was apparent from their higher tumorigenicity and their ability to disseminate and metastasize and to kill recipient mice more quickly. Upon histological examination of the local primary tumors a striking difference was noticed with regard to the degree of infiltration by host-derived mononuclear cells, mostly histiocytes. The non-metastasizing tumor Eb was heavily infiltrated while tumor ESb contained only a few of these cells. The differences between the tumor lines ESb and Eb are considered in the light of their possible relevance for metastases in general. The etiology of the two tumors is discussed in particular with respect to their relatedness.     

In vitro influence of gastrin,oestradiol and gonadotropin-releasing hormone on HCT-15 and LoVo human colorectal neoplastic cell proliferation

We set up in vitro several human colorectal neoplastic cell lines that we labelled “hormone-sensitive” (HS) in comparison to the original cell lines which appeared to be rather “hormone-insensitive” (HI). We used LoVo and HCT-15 human colorectal neoplastic cell lines and studied the influence of 17β-oestradiol (E2), gastrin and two gonadotropin-releasing hormone (GnRH) analogues, HRF and buserelin, on the proliferation of the HS and HI variants of the LoVo and HCT-15 cell lines. Cell proliferation was evaluated by a colorimetric assay, the MTT test. Our results show that E2, gastrin, HRF and buserelin did not induce a significant stimulatory influence on the HI variants of the LoVo and HCT-15 cells, i.e the cells that were cultured in a hormone-free 10% FCS-supplemented medium. In sharp contrast, the colorectal cells cultured for 30 passages in an E2 and/or gastrin + 1% FCS-supplemented medium showed a marked tropic response to E2, gastrin, HRF and buserelin. However, the HS variants of the HCT-15 cells appeared less sensitive to the two GnRH analogues than did the HS variants of the LoVo cells.     

Overlapping phenotypes of multidrug resistance among panels of human cancer-cell lines

In addition to P-glycoprotein (Pgp), 2 proteins related to multidrug resistance (MDR) have recently been described. The Multidrug-Resistance-associated protein (MRP) is one of the ATP-binding-cassette (ABC) transporters. The Lung-Resistance Protein (LRP) is the major component of human vaults, which are newly described cellular organelles and thought to mediate intracellular transport processes. Using immunocytochemical methods, we have examined the expression of MRP and LRP among panels of human cancer-cell lines not selected for drug resistance which have been previously characterized for expression of Pgp, and in vitro response to a variety of anti-cancer drugs. Expression of MRP and LRP was observed in 47/55 (87%) and 46/59 (78%) cell lines, respectively. Statistically significant correlations were observed between expression of each of these 3 proteins and in vitro sensitivity to at least one drug classically associated with MDR. LRP showed the greatest individual predictive value, which also applied to several non-classical MDR drugs. Co-expression of 2–3 MDR-related proteins was observed in 64% of the lines and was, in general, associated with high relative levels of drug resistance. Previously identified “classic” MDR lines as well as “pan-resistant” lines concurrently expressed all 3 MDR-related proteins. Some highly drug-resistant cell lines without detectable MDRI/Pgp were found to express relatively high levels of MRP and LRP. The high prevalence of MRP and LRP expression observed in this large set of cell lines, which have not been subjected to laboratory drug selection, suggests that MDR mechanisms associated with these proteins may be widespread in human malignancies. Moreover, the overlapping of these more recently recognized MDR phenotypes with Pgp-type MDR results in a complex phenotype, the understanding of which may be of importance in the development of new drugs and design of clinical treatment protocols, particularly those seeking to employ strategies to reverse the MDR phenotype. © 1996 Wiley-Liss, Inc.     

Use of an agar culture technique for establishing lymphoid cell lines from Marek's disease lymphomas

Lymphoblastoid cell lines derived from Marek's disease (MD) lymphomas have been established with difficulty by a number of workers. We have compared with conventional liquid culture methods the efficiency of a new technique for establishing lymphoid cell lines in which lymphoma cells were cultured initially in agar medium. Cells from 39/79 lymphomas gave rise to loose lymphoid colonies in the seeded agar after 7 days' incubation at 41°C. Two types of macrophage colony also developed. When lymphoid colonies in agar were transferred to liquid culture, 23/39 gave rise to permanent lymphoid cell lines, compared with 8/33 comparable cultures initiated in liquid medium. Twenty-nine new cell lines have been developed from Rhode Island Red, line 6 and line 7 chickens. All carry T-cell markers and the MD tumour-associated surface antigen (MATSA) and showed variable but low responsiveness to lectin mitogens. The new cell lines, when first established, consisted mainly of small, lymphocytoid cells but, after varying times, these changed into typical lymphoblastoid lines and an increased expression of an embryonic antigen was associated with this change. The lymphocytoid line cells were more slowly growing and densitydependent than were the lymphoblastoid cells, and lymphocytoid lines grew better at 41°C and lymphoblastoid lines better at 37°C. MD virus could be rescued from some of the lines but others appeared to be true nonproducers.