Lines of BPV-transformed murine cells that constitutively express influenza virus hemagglutinin.

We have developed and characterized several murine cell lines that constitutively express either the full-length, membrane-bound form of influenza virus hemagglutinin (HA) or a truncated version of the protein (HAsec) that lacks the carboxyterminal anchoring sequences and is secreted from cells. cDNAs encoding HA or HAsec were linked to the murine metallothionein-I promoter or the SV40 early promoter, and inserted into plasmids containing the transforming DNA fragment of bovine papilloma virus (BPV). The resulting vectors were introduced into three cultured lines of murine cells--C127, NIH3T3 and MME--either alone or in the presence of a plasmid that carries the aminoglycoside transferase gene of Tn5. The resulting lines of MME cells contained 1-5 copies of the vector in an integrated state and expressed low levels of HA (approximately 10(4) molecules/cell). In contrast, lines of C127 and NIH3T3 cells were obtained that express up to 5 X 10(6) molecules of HA per cell or secrete approximately 10(7) molecules of HAsec per cell per 24 h. Some of these cell lines carry multiple (30-200) copies of the vector in an integrated state; in others, the vector is propagated as unit-length episomes or as oligomers. Both the membrane-bound and secreted forms of HA expressed in these cell lines display a more extensive pattern of glycosylation than HA or HAsec synthesized in simian cells and they are transported to the cell surface more slowly. Pulse-chase experiments suggest that the step which limits the rate at which HA and HAsec travel down the secretory pathway occurs in the rough endoplasmic reticulum before the molecules are transferred to the Golgi apparatus. Using indirect immunofluorescence in combination with a cell sorter, we have shown that the level of expression of HA within cloned populations of producing cells can be variable. However, greater than 90% of the cells in certain cell lines display considerable quantities of HA on their surface, as judged by their ability to bind red blood cells in large numbers. We have taken advantage of the membrane fusion activity of HA to effect the fusion of erythrocytes to these cells and to deliver the contents of red cell ghosts into the cells' cytoplasm.     

A role for the thymic epithelium in the selection of pre-T cells from murine bone marrow

A rat thymic epithelial cell line IT45-R1 has been previously described as secreting soluble molecules that in vitro chemoattract rat hemopoietic precursor cells. The development of such an in vitro migration assay was based on the ability of cells to migrate across polycarbonate filters in Boyden chambers. In the present paper, by using the same strategy, we studied murine bone marrow cells capable of migrating in vitro toward IT45-R1 conditioned medium. The responding cells were shown to represent a minor bone marrow subpopulation characterized by a low capacity to incorporate tritiated thymidine in vitro (less than 10% of control). Moreover, this cell subset was considerably impoverished with respect to granulocyte-macrophage CFU (less than 7% of control) and pluripotent hemopoietic stem cells (less than 12% of control). Potential generation of T cells of donor-type in the lymphoid organs of irradiated recipients was measured by using C57BL/Ka Thy-1.1 and Thy-1.2 congenic mice. Thy-1.1 irradiated mice were injected intrathymically or intravenously with the selectively migrated cell subset of Thy-1.2 donor-type bone marrow cells. The use of an i.v. transfer route allowed us to show that these cells possess thymus-homing and colonization abilities. In a time-course study after intrathymic cell transfer, these migrated cells were able to generate Thy-1.2+ donor-type thymocytes represented by all cortical and medullary cell subsets in a single wave of repopulation from day 20 to day 30 after transfer, with a peak around days 23 to 25. The degree of repopulation closely resembled that seen with unfractionated bone marrow cells in terms of absolute numbers of donor cells per thymus (82% of control, 22 x 10(6) Thy-1.2+ cells) as well as in percent donor cells per thymus (105% of control). Thy-1.2+ cells were also detected in the lymph nodes and the spleens of reconstituted recipient mice. Taken together, these results support the idea that the supernatant of the established thymic epithelium IT45-R1 induces the migration of a murine bone marrow subset that contains hemopoietic stem cells already committed to the lymphoid lineage (i.e., pre-T cells).     

Retrovirus packaging cells based on 10A1 murine leukemia virus for production of vectors that use multiple receptors for cell entry.

10A1 murine leukemia virus can enter cells by using either of two different cell surface phosphate transport proteins, the gibbon ape leukemia virus receptor Glvr-1 (Pit-1) or the amphotropic retrovirus receptor Ram-1 (Pit-2). Glvr-1 and Ram-1 are widely expressed in different tissues, but the relative amounts of each are highly variable. We have developed retrovirus packaging cell lines based on 10A1 virus to take advantage of this dual receptor utilization to improve gene transfer rates in somatic cells of animals and humans, in which the relative levels of the two receptors are not always known. Optimization of the Env expression vector allowed the generation of packaging lines that produce helper-free vector titers up to 10(7)/ml. By interference analysis, we found that a 10A1 pseudotype retroviral vector can utilize Ram-1 for efficient entry into mouse, rat, and human cells and can utilize Glvr-1 for entry into mouse and human cells but not for entry into rat cells. The 10A1 pseudotype vector efficiently enters mouse cells by using Glvr-1, while entry into human cells is much less efficient. Thus, the 10A1 pseudotype packaging cells may be advantageous compared with the standard amphotropic packaging cells because vectors produced by the cells can use an additional receptor for cell entry. These packaging cells will also be useful to further explore the complicated pattern of receptor usage conferred by the 10A1 viral surface protein.     

Growth of tumor cells with different sensitivities for murine natural killer cells in young and adult athymic nude mice

Of four tumor cell lines, the murine YAC lymphoma, the human K562 lymphoma, and the human prostatic carcinomas PC3 and PC93, the susceptibility to murine natural killer (NK) cells as well as the tumorigenicity in young (3.5-4 weeks old) and in adult (8-10 weeks old) nude mice were studied. In young nude mice, which exhibited a lower level of NK cell activity than adult nude mice, the formation of solid tumors after inoculation of tumor cell suspensions occurred more frequently and with a shorter time lag than in adult animals. These effects were observed not only with the NK-sensitive YAC cells, but also with the relatively NK-insensitive PC3 and PC93 cells, indicating that also factors other than NK cell susceptibility may influence the growth of tumor cells in nude mice. Therefore, the use of young nude mice may enhance the rate of success of heterotransplantation of human tumors, regardless of the NK cell susceptibility of the tumor cells.     

Binding of iodinated multipotential colony-stimulating factor (interleukin-3) to murine bone marrow cells

Multipotential colony-stimulating factor (Multi-CSF or interleukin-3) was radioiodinated to high specific radioactivity (1-4 X 10(5) cpm/ng) with no detectable loss of biological activity and its binding to murine bone marrow cells and factor-dependent cell lines studied. Both the native glycosylated molecule purified from a cloned T-cell line (LB-3) and the purified non-glycosylated recombinant molecule produced by E. coli could be radioiodinated. Comparative binding studies with these derivatives demonstrated equal binding affinities and equal numbers of binding sites on various cell types indicating that carbohydrate moieties are not involved in the binding interactions. Binding of 125I-Multi-CSF to several factor-dependent continuous hemopoietic cell lines showed the presence of specific receptors on all cell lines, the receptor number per cell varying from 700 to 13,000 and the apparent dissociation constant from 400 pM to 1 nM. Specific binding of 125I-Multi-CSF was also observed to normal murine hemopoietic cells and the binding to murine bone marrow cells was studied in detail. Bone marrow cells showed 117-130 receptors per cell on average and an apparent dissociation constant of 126-233 pM. However, quantitative autoradiographic analysis indicated that receptors for 125I-Multi-CSF were not distributed randomly on bone marrow cells--nucleated erythroid and lymphoid cells were not labeled while essentially all neutrophilic granulocyte, eosinophilic granulocyte and monocytic cells were labeled. Moreover, in each of the labeled cell lineages grain counts (reflecting receptor number) decreased with increasing maturation and a small subpopulation of marrow cells (0.4-1.5% and including blast cells, monocytes, promyelocytes, and myelocytes) exhibited very high grain counts. The existence of such a subset of marrow cells raises the possibility of functional heterogeneity among marrow cells in their response to Multi-CSF.     

Cloning of murine gelsolin and its regulation during differentiation of embryonal carcinoma cells

The regulation of gelsolin levels during differentiation of the murine embryonal carcinoma cell line, PC-13, was investigated using nucleic acid and immunological probes. A cDNA clone, Mu-319, which contained the entire coding sequence for the cytoplasmic form of murine gelsolin was isolated using a polyclonal antibody. Gelsolin was detected in several cell lines but was not detectable in three undifferentiated embryonal carcinoma cell lines. Levels of gelsolin mRNA increased 10-fold during the differentiation of the murine embryonal carcinoma cell line, PC-13. Differentiation of PC-13 was accompanied by changes in cell shape, from small indistinct cells to large flat cells. The accumulation of gelsolin mRNA in PC-13 cells began 12-24 h after addition of the differentiation-inducing agents. In comparison, 2-5A-dependent RNase activity showed a 40-fold increase beginning after 24 to 36 h and c-fos mRNA were shown to increase about 9-fold beginning 36 to 60 h after induction of differentiation. The levels of gelsolin per se, as determined by immunoreactivity were also shown to increase with differentiation of PC-13 cells. These results suggest that gelsolin may play a role in the restructuring of actin filaments which accompanies the dramatic changes in cell shape during differentiation.     

The murine coronavirus mouse hepatitis virus strain A59 from persistently infected murine cells exhibits an extended host range.

In murine 17 Cl 1 cells persistently infected with murine coronavirus mouse hepatitis virus strain A59 (MHV-A59), expression of the virus receptor glycoprotein MHVR was markedly reduced (S. G. Sawicki, J. H. Lu, and K. V. Holmes, J. Virol. 69:5535-5543, 1995). Virus isolated from passage 600 of the persistently infected cells made smaller plaques on 17 Cl 1 cells than did MHV-A59. Unlike the parental MHV-A59, this variant virus also infected the BHK-21 (BHK) line of hamster cells. Virus plaque purified on BHK cells (MHV/BHK) grew more slowly in murine cells than did MHV-A59, and the rate of viral RNA synthesis was lower and the development of the viral nucleocapsid (N) protein was slower than those of MHV-A59. MHV/BHK was 100-fold more resistant to neutralization with the purified soluble recombinant MHV receptor glycoprotein (sMHVR) than was MHV-A59. Pretreatment of 17 Cl 1 cells with anti-MHVR monoclonal antibody CC1 protected the cells from infection with MHV-A59 but only partially protected them from infection with MHV/BHK. Thus, although MHV/BHK could still utilize MHVR as a receptor, its interactions with the receptor were significantly different from those of MHV-A59. To determine whether a hemagglutinin esterase (HE) glycoprotein that could bind the virions to 9-O-acetylated neuraminic acid moieties on the cell surface was expressed by MHV/BHK, an in situ esterase assay was used. No expression of HE activity was detected in 17 Cl 1 cells infected with MHV/BHK, suggesting that this virus, like MHV-A59, bound to cell membranes via its S glycoprotein. MHV/BHK was able to infect cell lines from many mammalian species, including murine (17 Cl 1), hamster (BHK), feline (Fcwf), bovine (MDBK), rat (RIE), monkey (Vero), and human (L132 and HeLa) cell lines. MHV/BHK could not infect dog kidney (MDCK I) or swine testis (ST) cell lines. Thus, in persistently infected murine cell lines that express very low levels of virus receptor MHVR and which also have and may express alternative virus receptors of lesser efficiency, there is a strong selective advantage for virus with altered interactions with receptor (D. S. Chen, M. Asanaka, F. S. Chen, J. E. Shively, and M. M. C. Lai, J. Virol. 71:1688-1691, 1997; D. S. Chen, M. Asanaka, K. Yokomori, F.-I. Wang, S. B. Hwang, H.-P. Li, and M. M. C. Lai, Proc. Natl. Acad. Sci. USA 92:12095-12099, 1995; P. Nedellec, G. S. Dveksler, E. Daniels, C. Turbide, B. Chow, A. A. Basile, K. V. Holmes, and N. Beauchemin, J. Virol. 68:4525-4537, 1994). Possibly, in coronavirus-infected animals, replication of the virus in tissues that express low levels of receptor might also select viruses with altered receptor recognition and extended host range.     

Deoxyglucose transport of uninfected, murine sarcoma virus-transformed, and murine leukemia virus-infected mouse cells

The initial rates of deoxy-D-glucose transport by cultures of growing and density-inhibited mouse embryo cells and lines of mouse cells transformed spontaneously or after infection by murine leukemia virus or murine sarcoma virus were investigated as a function of the deoxyglucose concentration. The apparent K_m for deoxyglucose transport was about the same for all types of cells (1–2 mM). The V_max of secondary cultures of mouse embryo cells decreased from 6 nmoles/10⁶ cells/minute for sparse cultures to less than 1 nmole/10⁶ cells/minute for density-inhibited cultures. The V_max was about the same whether estimated in monolayer culture or in suspensions of cells dispersed by treatment with trypsin. The V_max for deoxyglucose transport by the established cells, whether transformed spontaneously or by virus infection, was 4 to 25 times higher than that for density-inhibited mouse embryo cells and was independent of the cell density of the cultures. Deoxyglucose transport was competitively inhibited by Cytochalasin B, Persantin, glucose and 3-O-methyl-D-glucose and the apparent K_i values of inhibition were similar for the mouse embryo cells and the various cell lines. Similarly, the sensitivity of the glucose transport systems to inactivation by p-chloromercuribenzoate was about the same for all types of cells. The results suggest that the glucose transport system of the normal mouse embryo cells and the cells of the various established lines is qualitatively the same, but that the number of functional transport sites differs for the various cell lines and decreases markedly in mouse embryo cells with an increase in cell density of the cultures.     

Interleukin-3 induces proliferation but not lymphokine activated killer activity from human and murine mononuclear cells

Recombinant IL-3 (rIL-3) is a potent colony stimulating factor capable of stimulating early hematopoietic pluripotential progenitor cells and of supporting the differentiation of multiple cells. IL-3 has also been shown to have effects on mature, differentiated circulating cells including eosinophils and T cells. We evaluated the role of exogenous rIL-3 in the generation of cells with LAK activity from murine splenocytes and human bone marrow, spleen, unseparated PBMC and purified null cell preparations. rIL-3 was unable to generate lytic activity from any of these populations by itself and appeared to decrease LAK activity in bone marrow cultures containing high dose IL-2, (bone marrow derived cells (n = 3) with LAK activity for fresh tumor, mean lytic units(LU) 94.6 +/- 63.5 vs 32.8 +/- 44.8 for IL-2 and IL-2 plus IL-3 cultures, respectively p2 less than 0.05). Unlike previous reports testing murine cells, IL-3 priming and subsequent culture in IL-2 of human unseparated bone marrow cells or human or murine splenocytes, failed to generate long-term cultures with lytic activity. IL-3 did, however, induce a dose dependent stimulation of bone marrow and null cell preparations (mean null cell stimulation (3H Thymidine incorporation) with IL-3, 436 +/- 168 cpm vs 9802 +/- 9799 cpm, for 0 vs 10(3) units of IL-3, respectively n = 4, p2 less than 0.05). Furthermore, in bone marrow, unseparated PBMC and null cell cultures, the addition of rIL-3 generated characteristic large blastic appearing cells with prominent basophilic granules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Butanol-extractable and detergent-solubilized cell surface components from murine large cell lymphoma cells associated with adhesion to organ microvessel endothelial cells.

Metastatic variant cell lines of the murine RAW117 large cell lymphoma were used to study the cell surface components involved in syngeneic tumor cell/microvessel endothelial cell interactions. Poorly liver-metastatic parental RAW117-P cell line adhered to murine hepatic sinusoidal endothelial cell monolayers at significantly lower rates than the liver-selected, highly liver-metastatic RAW117-H10 line and both cell lines were poorly adherent to lung microvessel and bovine aorta endothelial cells. Viable, 2% 1-butanol-treated RAW117-H10 tumor cells formed fewer liver tumor nodules in experimental metastasis assays than untreated H10 cells and were significantly less adherent to murine hepatic sinusoidal endothelial cell monolayers. When 2% 1-butanol extracts of metabolically labeled or CHAPS detergent lysates of cell surface-labeled tumor cells were analyzed for their ability to bind to fixed microvessel endothelial cell monolayers, quantitative differences were found in the extractable tumor cell surface components that bound to the different organ-derived microvessel endothelial cells. Cell surface components (1-butanol extractable), of Mr approximately 85,000-90,000 and approximately 37,000-40,000 bound to hepatic sinusoidal endothelial cell monolayers to a greater extent than to murine lung microvessel endothelial or bovine aortic endothelial cell monolayers, whereas tumor cell surface components of Mr approximately 45,000, approximately 33,000, and approximately 25,000 bound similarly to endothelial cells regardless of origin. The results suggest but do not prove that tumor cell/endothelial cell adhesion involves multiple tumor cell surface components, some of which commonly bind to various endothelial cells and others for which binding may be dictated by the tissue origin and type of endothelial cell. Particular RAW117 butanol-extractable cell membrane components were associated with tumor cell-endothelial cell adhesion, and these components could be responsible, in part, for the preferential adhesion of RAW117 cells to liver sinusoidal endothelial cells and metastasis to liver.     

Analysis of androgen-sensitivity in rat prostate X mouse kidney cell hybrids

Variant androgen-sensitive cell lines were produced by fusing freshly isolated epithelial cells from the rat ventral prostate with a line of murine renal tumor (RAG) cells. The properties of the cloned lines of the prostate X RAG hybrids can be summarized as follows: (1) the modal chromosome number of the hybrid cell lines ranged from 68 to 176; (2) the cells had doubling times of 7.6-49.5 h; and (3) epitheloid, ameboid and intermediate morphologies were observed among the various lines. The proliferative response of various hybrid lines to treatment with 10 nM 5 alpha-dihydrotestosterone was used to classify the hybrids as either very sensitive (greater than 40% reduction in cell doubling time), sensitive (greater than 10% reduction in doubling time) to androgens, or insensitive (less than 10% reduction in doubling time) to androgens. There was no direct relationship between the androgen-sensitivity of the cells and their androgen receptor content, suggesting that these variant cell lines may be useful for the study of the genetic factors involved in cellular responses to androgens.     

Constitutive versus cell cycle regulation of c-myb mRNA expression correlates with developmental stages in murine B lymphoid tumors.

The expression of c-myb mRNA is differentially regulated in murine B lymphoid tumors such that B cell lymphomas and plasmacytomas contain significantly less c-myb mRNA than pre-B cell lymphomas. To examine the low level of c-myb mRNA expression in the murine B cell lymphoma cell line BCL1, nonessential amino acid starvation was used to block these cells in a G1 state. When BCL1 cells were released from this block, a 7- to 10-fold increase in c-myb mRNA was detected in late G1 and S phase cells relative to that detected in exponentially growing BCL1 cells. This increase was not inhibited by aphidicolin. To determine whether cell cycle regulation of c-myb mRNA expression occurred during exponential growth in both murine pre-B cell lymphoma and B cell lymphoma cell lines, elutriation was used to separate exponentially growing cell populations. An increase in c-myb mRNA expression was seen in late G1 and S phase fractions from B cell lymphoma cell lines. In contrast, c-myb mRNA levels remained constant in elutriation fractions isolated from pre-B cell lymphoma cell lines. Expression of c-myb mRNA was not detected in exponentially growing or in Go serum-stimulated murine fibroblasts. These results indicate that constitutive vs cell cycle regulation of c-myb mRNA expression is related to the state of differentiation in murine B lymphoid tumors and suggest that a switch in regulation may occur during normal B cell development.     

Cloning of murine and rat vascular cell adhesion molecule-1.

Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the integrin very late antigen 4 (VLA4). We have cloned the cDNAs for both murine and rat VCAM1 from endotoxin-treated lung libraries. Both sequences encode proteins with seven extracellular Ig-like domains, which show 75.9% and 76.9% identity, respectively, with human VCAM1. Both murine and human cell lines show VLA4-dependent binding to COS cells transiently expressing murine and rat VCAM1. Two mAbs, M-K/1 and M-K/2, which recognize an antigen on murine bone marrow stromal cell lines, bind to murine VCAM1 expressed in COS cells and block VCAM1-dependent adhesion, confirming that these mAbs recognize murine VCAM1.     

A phenyl-beta-galactoside-specific monoclonal antibody reactive with murine and rat NK cells

The phenyl-beta-galactoside (phi-beta-gal)-specific monoclonal antibody (mAb) 49H.8 cross-reacts with the terminal disaccharide structure of the asialo GM1 (AGM1) molecule. It was found to react with phi-beta-gal determinants on murine and rat splenic natural killer (NK) cells, as measured by complement depletion studies. Flow cytometric analysis identified the antigen on two IL 2-dependent cloned murine NK cell lines and the rat large granular lymphocyte leukemia RNK. We have compared the 49H.8 reactivity to that of anti-AGM1 antisera (alpha-AGM1) on NK cells and a panel of NK related killer cells, including bone marrow-derived killer cells, lymphokine-activated killer cells (LAK), and anomalous killer cells (AK). We found that the 49H.8 specificity closely paralleled that of alpha-AGM1. When tested against Con A-reactive T cells, the 49H.8 mAb was less reactive than the alpha-AGM1, indicating that it may be a more specific marker for splenic NK populations than the alpha-AGM1.     

Knockdown of antizyme inhibitor decreases prostate tumor growth in vivo

The endogenous protein antizyme inhibitor (AZI) is a potential oncogene which promotes cell growth by both inhibiting antizyme (AZ) activity and releasing ornithine decarboxylase (ODC) from AZ-mediated degradation. High levels of ODC and polyamines are associated with numerous types of neoplastic transformation, and the genomic region including AZI is frequently amplified in tumors of the ovary and prostate. To determine whether AZI functionally promotes prostate tumor growth, we made PC3M-LN4 (human) and AT6.1 (rat) cancer cell lines stably expressing shRNA to knockdown antizyme inhibitor 1 (AZI). AZI knockdown was confirmed by western blot, quantitative real-time PCR, and immunofluorescence. To examine the ability of these cells to form tumors in vivo, 1 × 10(6) cells were injected subcutaneously into nude mice either with (PC3M-LN4) or without (AT6.1) Matrigel. Tumor growth was measured two times per week by caliper. We found that cells in which AZI levels had been knocked down by shRNA formed significantly smaller tumors in vivo in both human and rat prostate cancer cell lines. These results suggest that not only does AZI promote tumor growth, but also that AZI may be a valid therapeutic target for cancer treatment.     

Antibody-dependent cellular cytotoxicity against murine leukemia viral antigens: studies with human lymphoblastoid cell lines and human peripheral lymphocytes as effector cells comparing rabbit, goat, and mouse antisera.

A thymic lymphoblastoid cell line derived from a New Zealand Black mouse produces murine leukemia virus (MuLV) and was used as a target in model systems for the in vitro study of antibody-dependent cellular cytotoxicity (ADCC). Several human lymphoblastoid cell lines were investigated as potential effector cells. The most promising (Raji cells) bound to antibody-coated target cells but caused only modest levels of ADCC at 25:1 effector-to-target cell ratio with substantial lysis in the absence of antiserum. Human peripheral lymphocytes were active as effector cells in ADCC at a 5:1 ratio and produced no lysis in the absence of antibody. These cells were used to demonstrate that high dilutions of rabbit antisera to MuLV antigens p30, p15, p12, and p10 were capable of mediating lysis of MuLV-producing target cells but not of a virus-negative murine cell line. A murine antiserum to Thy 1.2 and three caprine antisera to MuLV antigens that were active in complement-mediated cytotoxicity functioned poorly in inducing ADCC; however, rabbit antisera to similar antigens were 16- to 512-fold more efficient in cell-mediated than in complement lysis. The inefficiency of goat antisera was not due to shedding of cell surface antigens or generation of blocking factors but rather to lack of lytic interaction of antibody-coated targets with the effector cells.     

Estrogen and progesterone receptors in some human myeloma cell lines and murine hybridomas

The control of immune responses by sex hormones is well documented but the effect of sex hormones on lymphoid cell subsets is poorly understood. We have investigated the expression of receptors for androgens (AR), estradiol (ER) and progesterone (PR) by human cell lines of the B lymphocyte lineage and by murine myeloma or hybridomas. AR, ER and PR were determined by cytosol and nuclear binding assays. Eleven human lymphoblastoid cell lines obtained by in vitro infection of blood or tonsil B cells with Epstein-Barr Virus (EBV) B95, did not express AR or ER. Similarly, 10 Burkitt's lymphoma cell lines were AR, ER and PR negative with the exception of the pre-B RAJI cells which bear AR. Among 13 cell lines derived from patients with multiple myeloma none expressed AR but five were found to bear ER (20-164 fmol/mg DNA or 5-10 fmol/mg protein). Four of the latter group also bear PR (86-450 fmol/mg DNA). Two mouse hybridomas out of seven tested were ER and PR positive. The MOPC 315 myeloma expressed ER but not PR. The possible functional role of these sex hormone binding sites in cell proliferation and immunoglobulin secretion deserves further investigation.     

Antiproliferative activity of lichen extracts on murine myeloma cells

In the present study we report some preliminary results concerning the evaluation of antiproliferative activity on murine myeloma cells (P3X63-Ag8.653) of crude extracts of two common lichen species, Evernia prunastri and Xanthoria parietina. The results were evaluated by means of the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test, which is commonly used to assess the activity of living cells through mitochondrial dehydrogenases. They indicated that extracts of E. prunastri had no effect, while those of X. parietina significantly affected murine myeloma cell proliferation, with a reduction down to 75% for methanolic extracts. This opens perspectives for deeper investigations extended also to other mammalian cell lines.