Five measles virus antigens demonstrated by use of mouse hybridoma antibodies in productively infected tissue culture cells

Summary Mouse hybridoma antibodies against 5 different structural components of measles virus were used in immune fluorescence tests to characterize the appearance of viral antigens in productively infected cell cultures. The antibodies employed in the tests reacted specifically with the hemagglutinin (H, 79K), polymerase (P, 72K), nucleocapsid (NP, 60K) hemolysin-fusion factor (F, 41+20K) and matrix (M, 36K) proteins. Syncytia formed in lytically infected cultures and single isolated cells in persistently infected cell cultures were both examined.Antibodies against NP and P proteins stained cytoplasmic inclusions varying in size from small dots to more confluent masses, frequently in a perinuclear position. Nuclei of infected cells contained exclusively NP antigen. Antibodies to envelope components—H, F and M—stained cytoplasmic membrane structures and also gave a granular cytoplasmic staining, especially in syncytia. Although all persistently infected cells produced NP antigen and the associated P component, they had a restricted capacity to produce demonstrable amount of envelope antigens. The occurrence of cells containing envelope antigens varied between about 50 and 5 per cent with H and F antigens giving the highest and lowest frequence values, respectively. It is proposed that a restricted capacity of cells to produce biologically active fusion protein is a prerequisite for maintaining a persistant infection in actively dividing cellsin vitro.With 4 FiguresThis work was supported by grants from the Swedish Medical Research Council (Project no. B81-16X-00116-17A) and the U.S. Public Health Service (NS 16463).     

Retrospective study of Western blot profiles in immune sera of natural dengue virus infections

The Western blot (WB) assay was used to determine dengue virus antibodies present in human immune sera arising from recent primary and secondary dengue virus infections in Singapore. Cell lysates of dengue-2 virus-infected C6/36 and Vero cells were used. Antibodies directed against structural proteins of dengue-2 virus including envelope (E, gp60/50), capsid-premembrane (C-PrM, gp35), and premembrane (PrM, gp20) were detected, with antibody against envelope protein being most dominant. Similar WB profiles were detected in both primary and secondary dengue virus infections. The reactivity rate of antibodies to dengue-2 virus proteins was higher in infected Vero cell lysate than in infected C6/36 cell lysate, with the exception of antibodies to nonstructural proteins of NS1 and NS3, which were detected predominantly in infected C6/36 cell lysate. More than 75% of "normal" individuals (with no complaint of recent dengue virus infection) examined had low levels of dengue virus antibodies, but all presented with similar WB profiles as patients with recent dengue virus infections. This finding reflects a high seroprevalence of dengue virus infections and the long lasting nature of E, C-PrM, and PrM antibodies. Results from this study indicate that in natural dengue virus infections, native E, C-PrM, and PrM antigens of dengue virus are immunogenic and elicit long-lasting antibodies.     

Detection of virus antigens in Swiss albino mice infected by milk-borne mouse mammary tumour virus: the effect of age, sex and reproductive status. I. Localization by immunofluorescence of four antigens in mammary tissues and other organs.

The indirect immunofluorescence technique was used to investigate the expression of virus antigens in a Swiss albino mouse strain infected by mouse mammary tumour virus (MMTV). The antisera used were monospecific for: the glycopolypeptides gp160 and gp47, and the internal polypeptides p28 and p8. In mice infected with milk-borne MMTV, all four antigens were detected in the mammary gland during the first pregnancy and then throughout life, and in mammary tumours. Antigen gp160, located at the secretory border of all glandular cells, is shared by the plasma membrane of the mammary cell, whether virus-producing or not, and by the virus envelope, as shown by the use of absorbed antisera. The other three antigens are virus-specific. Anti-p47 serum stained the secretory border of cells whereas the fluorescence related to p28 and p8 consisted of a few large intracytoplasmic spots. The specific antibodies for p28 and p8 were only absorbed by disrupted virions, confirming that these polypeptides are internal antigens. With serial sections of glands from pregnant and old female mice, the alveoli stained with anti-p28 or anti-p8 serum were found to react also with anti-gp47 serum. However, during lactation, the presence of p28 and p8 could not be detected in the alveoli stained with anti-gp47 serum. Thus, infected alveoli express virus antigens differently. In the mammary glands of all mice studied, some alveoli did not display any staining when the latter three antisera were used. The specific antigenic pattern, maintained in differentiated mammary tumours, became a diffuse and unlocalized staining after transformation into the undifferentiated state. With the exception of the kidney, where gp47 was found in the glomeruli, all other organs of the mouse did not stain with any of the antisera. The presence of gp47 in the glomeruli, probably related to immune-complex deposits, increases with the age of the mouse and is most noticeable in tumour-bearing females. In mice free of milk-borne MMTV, examined during the first week of lactation, antigen gp47 could not be detected. Internal antigens p28 and p8 were detected in nearly all mammary cells as numerous small cytoplasmic dots. This suggests a limited expression of an endogenous virus genome; this expression would be modified when milk-borne virus is produced.     

Peptide antisera targeted to a conserved sequence in poliovirus capsid VP1 cross-react widely with members of the genus Enterovirus.

Rabbits were immunized with synthetic peptides derived from an immunodominant region of the VP1 capsid protein of enteroviruses. This region shows a high degree of homology among all sequenced members of the genus. Peptide-induced antisera were used for immunoperoxidase staining of cell cultures infected with 41 different serotypes of enterovirus. Specific cytoplasmic staining was readily seen in all but two cases. Echovirus type 22 was previously known to differ genetically from the rest of the enteroviruses, and hence, a negative result was expected. Surprisingly, one of the tested serum samples reacted with echovirus 22-infected cells. Coxsackievirus A7-infected cells could be reliably stained with only one of the tested serum samples. For the remaining 39 serotypes, scattered infected cells resulting from 1 to 2 days of incubation with diluted inocula were easily scored as positive before the cytopathic effect became visible. The same antibodies were also used in a sandwich-type enzyme immunoassay to demonstrate poliovirus antigens in cell extracts as early as 3 h after a high-multiplicity infection. These antibodies are candidates for enterovirus group reagents, being potentially useful in both the laboratory diagnosis of enterovirus infections and research on enterovirus-host interactions.     

Nuclear immunofluorescence in porcine kidney cells infected with Japanese encephalitis virus.

Acetone-fixed porcine stable kidney (PS) cells infected with Japanese encephalitis (JE) virus were stained in indirect fluorescent antibody (FA) assay with anti-JE virus monoclonal (MoAb) and polyclonal (immune PF) antibodies. First positive immunofluorescence (IF) occurred in the cytoplasm with MoAb Hs-1 (anti-envelope, JE-specific) and immune PF after 7 hr post-infection (p. i.); it became prominent by 15 hr to 48 hr (maximum) when cells reacted strongly also with MoAb Hx-3 (flavivirus crossreactive epitope). In addition, 15 to 20% of the infected cells, which revealed positive cytoplasmic IF, showed intranuclear IF with Hs-1, Hx-3, and immune PF by 20 to 24 hr p.i. By 48 hr, the intranuclear IF was not observed or became diminished. These observations indicate that the JE virus specific epitope Hs-1 appeared first followed by the flavivirus cross-reactive epitope Hx-3. Nuclei of the infected cells seem to play some role in the replication of JE virus.     

Serological and immunochemical characterization of monoclonal antibodies to Toxoplasma gondii.

The fusion of mouse NS-1 myeloma cells with spleen cells from mice chronically infected with Toxoplasma gondii resulted in eight clones of hybridomas producing monospecific antibodies against membrane or cytoplasmic antigens of Toxoplasma tachyzoites. One of the antibodies to a cytoplasmic determinant was an IgM; the others directed to membrane or cytoplasmic antigens belonged to the IgG2 or IgG3 isotypes. Antibodies of clones 1E11 (IgG3), 2G11, and 3E6 (IgG2) directed to membrane antigens, bound complement and were reactive in the complement-dependent cytotoxicity assay of Sabin-Feldman. These IgG2 antibodies were strongly agglutinating to parasites, whereas the IgG3 was relatively weak. Another IgG2 antibody (5B6), possibly recognizing a shared antigen of membrane and cytoplasm, exhibited a low titre in the cytotoxicity assay as well as in the agglutination assay. Two other antibodies to membrane antigens (2B7 and 2F8) as well as an antibody to a cytoplasmic antigen (3G3) did not bind complement and did not cause agglutination. The pattern of parasite staining produced by monoclonal antibodies to membrane antigens in an IFA test was different from that of polyvalent antisera. A strictly localized or 'beaded' staining was observed, as well as a smooth, rim fluorescence. Toxoplasma tachyzoites were surface radio-iodinated and the solubilized membrane proteins were immunoprecipitated with monoclonal antibodies and analysed by two-dimensional polyacrylamide gel electrophoresis. Two independently arising monoclonal antibodies to membrane antigens (2G11 and 3E6) consistently precipitated both the solubilized 35,000 and 14,000 mol. wt proteins, while 1E11 precipitated the 27,000 mol. wt protein.     

Distribution by immunofluorescence of viral products and actin-containing cytoskeletal filaments in rubella virus-infected cells

Summary Rubella virus (RV)-host cell interactions were examined by indirect immunofluorescence staining using antibodies to viral products and cytoskeletal components as probes. The patterns of immunofluorescence observed with human convalescent sera indicated that in infected Vero cells RV-specified proteins were distributed throughout the rough endoplasmic reticulum with some possible accumulation in the region of the Golgi complex. Viral RNA synthesis, detected with anti-double stranded RNA, appeared to be confined to small, intensely stained foci irregularly distributed in the cytoplasm. When cells were infected at a higher multiplicity, these foci appeared to aggregate into linear arrays. Infection with RV had a profound effect on the organization of actin in both Vero and BHK 21 cells, as shown by anti-actin antibodies. Actin microfilaments were observed to disintegrate progressively into amorphous aggregates of apparently monomeric actin as the infection proceeded. Because of the role actin microfilaments may play in cell mitosis it is postulated that this effect may be related to the inhibition of cell division reported to be associated with the congenital rubella syndrome.With 3 Figures     

Serological studies of Actinomyces israelii by crossed immunoelectrophoresis: taxonomic and diagnostic applications.

Crossed immunoelectrophoresis (CIE) with intermediate gel was applied to the serological analysis of Actinomyces israelii to develop a test with high efficiency in the laboratory diagnosis of human actinomycosis and classification of A. israelii. Recently developed standard antigen-antibody systems for A. israelii by CIE were used as reference. The reference systems were based on standard preparations of cytoplasmic and whole cell-associated antigens of A. israelii and a standard immunoglobulin G pool purified from rabbit antisera to formalin-treated whole cells and cell lysates of A. israelii. The specificity of the standard antigens for A. israelii was evaluated in CIE studies by screening for antibodies to components of the antigens in rabbit antisera raised against related bacteria. The standard system for A. israelii based on cytoplasmic antigens formed species-specific precipitins whereas antisera raised against A. naeslundii and/or Propionibacterium acnes precipitated components of the other standard antigens. As a result of these analyses, the standard system for A. israelii based on 10 cytoplasmic antigens was used as reference for CIE studies to detect humoral antibodies to A. israelii in sera from nine patients with actinomycosis. All the sera from the patients formed at the time of diagnosis one or more precipitins in terms of the 10 reference precipitins. Up to five precipitins were found in single sera. Follow-up studies covering a period of one-half year after treatment showed a gradually decreased precipitin response in the course of time. In control sera from patients with newly diagnosed tuberculosis, nocardiosis, deep Candida infection, and aspergillosis, and in sera from healthy blood donors, no antibodies were detected with specificity for the reference antigens.     

An electron and immunoelectron microscopic study of dengue-2 virus infection of cultured mosquito cells: Maturation events

Summary The maturation process of dengue-2 virus in C6/36 mosquito cells was studied by electron microscopy at 12, 16, 24, 48, and 78 hours postinoculation (p.i.) and by immunoelectron microscopy at 48 and 78 hours p.i. Maturing virions appeared within cytoplasmic vacuoles and on the surface of infected cells from 24 hours p.i. onward in close topographical relationship to the dense particles that occurred concurrently in the cytoplasm. The dense particles measured 25 to 35 nm in diameter; the mature virions measured 50 to 55 nm in diameter, with a dense core measuring 30 to 35 nm in diameter covered by a 10 nm-thick membrane envelope. The morphological observations indicated that the dense particles were dengue nucleocapsids assembled in the cytoplasm and that they apparently budded into the vacuolar lumens and the extracellular space at the vacuolar and plasma membranes, acquiring membrane envelopes and becoming mature virions in the process. The virions that budded into the vacuolar lumens were released extracellularly by exocytosis. In the samples tested with dengue-2 polyclonal antibodies, intense immunostaining occurred at the sites of virus budding on the cell surface; host cell membrane and cytoplasm adjacent to the budding virions stained less intensely. In the samples tested with a dengue-2 monoclonal antibody specific for the envelope glycoprotein, budding virions stained rather exclusively, with no staining occurring in adjacent host membrane or cytoplasm.With 10 Figures     

Antibody- and complement-mediated lysis of HIV-infected cells and inhibition of viral replication

HIV-1-positive antisera were tested for their ability to lyse HIV-1-infected cells in the presence of active complement. Cytolytic effects caused by sera derived from infected humans were slower than those observed with sera from immunised chimpanzees. Lytic but also negative sera were found among HIV-1-infected asymptomatic men as well as among clinical AIDS cases. Human antisera that lysed infected cells reacted similarly irrespective of whether the complement was heterologous or autologous. Analysis of complement-mediated lysis using defined antisera against recombinant HIV-1 env or core antigens suggested that gp160/gp120 and p24 can act as target antigens for an antibody- and complement-mediated cytolysis of infected cells. Complement alone reduced the spread of HIV-1 infection in CD4+ cells and the ability of HIV-1 and HIV-2 to form plaques in CD4-transfected HeLa cells. Co-operative effects of specific antibodies and complement were the most effective in inhibiting HIV infections.     

Ultrastructural localization of viral antigen in nuclear inclusions of cytomegalovirus infected guinea pig cells

Summary Intranuclear localization of viral antigens in guinea pig cytomegalovirus (GPCMV) infected guinea pig embryo (GPE) cells was investigated by cross-reactive indirect immunoperoxidase and immunoferritin techniques utilizing guinea pig antisera to GPCMV. Following primary fixation with 4 percent paraformal-dehyde, a brief treatment of infected cells with 0.25 percent trypsin was found to enhance penetration of antibodies and the conjugates. Ferritin or horseradish peroxidase conjugated goat anti-rabbit IgG was used as a secondary antibody that cross reacted with guinea pig immunoglobulins in order to reduce non-specific immunochemical reactions. Using light microscopy following immunoperoxidase staining, GPCMV antigens in an intranuclear location were not discernable when the infected cells were stained without pretreatment with trypsin, however intranuclear GPCMV antigens could be visualized after the fixed cells were treated with trypsin for 2–4 minutes prior to addition of the antiserum. Electron microscopic examination following indirect immunoferritin staining revealed viral antigens localized on viral capsids and on scattered electrondense amorphous matrices but not on the surrounding tubular structures or fibrils. The possibility that tubular structures may be a host cell product produced in response to GPCMV infection is discussed.With 3 Figures     

Immunofluorescent detection of surface antigens specific to T and B lymphocytes in the chicken

Rabbit antisera specific for chicken T and B cells as judged by surface immunofluorescent staining have been raised. Specificity was established by the staining of thymus and bursal cell suspension and by the effects of thymectomy and bursectomy on the staining of peripheralized lymphocytes. Furthermore, double labeling experiments showed that anti-T and anti-B sera reacted with different populations of blood lymphocytes. Comparable numbers of cells in blood and spleen stained for B and light chain determinants. No evidence for “null cells” was obtained. There was little change in the percentage of cells staining in the various lymphoid organs from 4 days to 12 months of age. The thymus contained approximately 7 % B cells, although no T cells were demonstrable in the bursa. One antiserum showed only thymocyte specific antibodies not reacting with peripheral T cells. The specific B and T markers seem to be acquired during differentiation within the appropriate central lymphoid organ. Demonstrable surface immunoglobulins appear later in ontogeny than the B antigens. The majority of cells bearing the B marker in bone marrow were large cells lacking surface light chain determinants.     

Detection of some dengue-2 virus antigens in infected cells using immuno-microscopy

Summary Immunoelectron microscopy was used to detect the distribution of some dengue-2 virus proteins in infected Vero andAedes albopictus (C 6/36) cells. It was found that the envelope protein (GP 60) was located in clumps on the surface of plasma membrane, and accumulated very little in the infected cytoplasm. However no envelopment of dengue-2 virus nucleocapsids through the plasma membranes was observed. In contrast, the NS 3 (P 67) protein was distributed throughout the whole cytoplasm. No specific association of this protein with the proliferated virus-induced structures was seen. The NS 1 (GP 46) protein showed almost similar distribution as the NS 3 but its quantity appeared to be lower. The NS 3 and NS 1 distribution patterns observed supported the results of immunofluorescent staining but results on E protein were not consistent in both methods.     

Detection of flavivirus antigens in purified infected Vero cell plasma membranes.

The types of Kunjin virus-specified proteins present in purified Vero cell plasma membrane were studied. Immunofluorescence of unfixed Kunjin virus-infected whole cell monolayers, indicated that two structural proteins (envelope and prM) and three non-structural proteins (NS1, 3 and 5) were found at the plasma membrane. There was no obvious progressive accumulation of the observed antigens over the time periods between 8 to 24 h p.i. Thus SDS-PAGE analysis was performed using purified radiolabelled Vero cell plasma membranes. From the protein profiles, all five antigens detected by immunofluorescent staining were also present. In addition, two smaller molecular weight non-structural proteins NS4B and NS2B were also observed. Generally, all the non-structural proteins found in the purified plasma membranes were of the same molecular weights as those found in infected whole cell lysate. Interestingly, both the structural proteins, i.e., envelope (E) and prM proteins in the plasma membrane sample were of higher molecular weights as compared to the counterparts in the infected whole cell lysate. The envelope protein of purified extracellular Kunjin virus was also lower in molecular weight compared to the same protein in the plasma membrane.     

初次及再次感染HCV后不同功能区抗体的研究

１４例初次ＨＣＶ感染者及１１例再次ＨＣＶ感染者系列血清系在无法检测ＨＣＶ时留存的２８９位手术受血者系列血清中筛选获得。系列血清包括受血前、受血后不同时期收集的血清。回顾性地研究其不同功能区抗体出现的动态变化，抗Ｃ与抗ＮＳ３抗体有早期诊断价值。抗ＮＳ３、抗Ｃ、抗ＮＳ５及抗ＮＳ４抗体在ＨＣＶ感染后系列血清中检出率分别为８４．７６％、７９．２７％、７２．５４％和６８．３９％。感染过程中各区抗体可以全部出现、部分出现或单独出现抗Ｃ、抗ＮＳ３及抗ＮＳ５抗体，未发现单独含抗Ｅ、抗ＮＳ１及抗ＮＳ４区抗体的血清。抗Ｅ、抗ＮＳ１及抗ＮＳ４抗体消失较早。研究表明：以ＨＣＶＣ区、ＮＳ３区及ＮＳ５区编码的优势抗原包被的ＥＬＩＳＡ抗体诊断试剂盒将提高ＨＣＶ的诊断。     

Alteration of virus entry mode: a neutralisation mechanism for Dengue-2 virus

Polyclonal antibodies derived from dengue virus immune sera and 3H5 monoclonal antibody showed potent neutralisation effect on dengue-2 virus in the plaque reduction neutralisation assay. This study demonstrated that antibodies present in immune human sera and 3H5 monoclonal antibody neutralised dengue-2 virus by altering the virus entry pathway into cells. In the presence of neutralising antibodies, dengue-2 virus was endocytosed by LLC-MK2 cells. The endocytosis process involved ruffling of antibody-coated virions by cellular pseudopodia and invagination of cell membrane. This mode of entry is atypical as compared to direct fusion of dengue-2 virus with cell membrane in the absence of antibody. The virions were internalised in the form of virion-antibody complexes consisting of single or clumps of virions. After 3 minutes of incubation, neutralised virions were detected in cellular vesicles, and signs of intra-endosomal penetration into cytoplasm were not evident even after a prolonged incubation of 10 minutes, suggesting that viral uncoating was compromised. Vesicle-bound virions were no longer detected after 20 minutes of incubation. In addition, no sign of viral replication was detected in cells infected with "neutralised" virions by immunofluorescence assay. This indicated that internalised virions had been degraded leading to abortive infection. In conclusion, antibodies present in 3H5 monoclonal antibody and human immune sera rendered dengue-2 virus non-infective by neutralising the viral fusion site and causing alteration of viral entry mode. Antibodies in immune sera but not 3H5 monoclonal antibody also exerted minimal inhibitory effect on virus binding and internalisation.     

Some characteristics of three porcine adenoviruses

Summary Three strains of cloned porcine adenoviruses (25RCl, 6618Cl and A47C1) were shown to be serologically distinct in cross neutralization tests with specific antisera prepared in rabbits. They were not neutralized by reference antisera against the known human adenoviruses. Neutralizing antibodies to each of the three serotypes were demonstrated in samples of pig serum. Guinea pig, human 0, rat, monkey and mouse red cells were haemagglutinated by strain 25RCl, but fowl, ox, pig, rabbit and sheep cells were not haemagglutinated. Strains 6618Cl and A47Cl did not haemagglutinate these cells. Specific rabbit antiserum and normal pig sera contained HI antibodies to strain 25RCl adenovirus. Each of the three strains was resistant to ether and pH 4.0 buffer and growth in PK cells was inhibited by FUDR. In infected PK cells stained with haemotoxylin and eosin, typical basophilic central nuclear masses were produced by each strain of porcine adenovirus. In PK cells infected with strain 6618Cl multiple eosinophilic intranuclear inclusions in some cells were a prominent feature of the CPE. The provisional designation of strains 25RCl, 6618Cl and A47Cl as porcine adenovirus types 1, 2, and 3, respectively, is proposed.     

Histamine immunocytochemistry: a new method for detection of basophils in peripheral blood

We report that basophils in peripheral blood can be stained using histamine immunocytochemistry. The staining is based on the fixation of leucocytes with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (CDI) and the subsequent incubation of these cells with antisera raised against histamine conjugated to different carrier proteins using CDI. The staining appears to be specific for basophils and stained cells can be examined using both fluorescence microscopy and flow cytometry. In addition, histamine immunocytochemistry can be combined with conventional immunocytochemistry by incubating leucocytes with antibodies to cell surface antigens prior to or following fixation of the cells with CDI. Thus, histamine immunocytochemistry may be a valuable tool in future studies of human basophils.     

Surface-reactive antibodies to human adrenal cells in Addison's disease.

Organ-specific surface-reactive antibodies to viable human adrenal cell suspensions from adult or fetal glands were detected by indirect immunofluorescence (IFL) in 24 out of 28 idiopathic Addison's disease sera with adrenal cytoplasmic antibodies. Cell-surface reactions were also present in nine out of 10 cases of polyendocrine autoimmune disorders without overt adrenal failure but possessing adrenal cytoplasmic antibodies. None of 18 Addisonian patients, 25 cases with other autoimmune disorders and 10 normal individuals, all negative for adrenal cytoplasmic antibodies, showed positive surface reactions on viable cells. When the surface IFL was done on established monolayers, the positive sera gave variable staining suggesting that more than one antigen may be expressed under different conditions. These results suggest that adrenal-specific 'microsomal' antigens are also represented on the plasma membrane, and support the hypothesis that organ-specific autoantibodies reacting with the surface of living target cells may have a pathogenic role in the development of autoimmune adrenalitis.     

Cytotoxic Antibody Response to Tumors Induced in Adult and Newborn Rabbits by Fibroma Virus

Cells cultured from tumors induced in rabbits by inoculation of fibroma virus possessed virus-specific cell surface and cytoplasmic antigens. Tumor cell cultures were capable of a limited number of cell divisions before degenerating. Employing the (51)Cr-release test and the microcytotoxicity test, it was demonstrated that sera from rabbits with regressed fibroma tumors contained antibodies cytotoxic for cells infected in culture with fibroma virus. These sera were only weakly cytotoxic for cultured fibroma tumor cells. In addition, newborn rabbits bearing progressively growing tumors had serum antibodies cytotoxic for cells infected with fibroma virus in culture but not for fibroma tumor cells. Immunofluorescence studies also showed that the reaction of immune serum with the surface antigens of fibroma tumor cells was markedly weaker than with the surface antigens of cells infected with virus in culture. Furthermore, membrane cytotoxicity and immunofluorescence reaction were significantly reduced when cells were tested after prolonged incubation or cell division, or both, following infection with fibroma virus. The failure of tumors to regress in newborn rabbits in spite of the presence of cytotoxic antibodies is discussed in respect to a possible reduction or alteration of virus-specific surface antigens on proliferating tumor cells.