Samuel Salzberg

The response of normal and malignant cells to ultrasound in vitro

The effect of ultrasonic irradiation on the viability of normal and tumor cell cultures derived from human and mouse origins was investigated. The cells were irradiated with a frequency of 2 MHz and intensity of 0.33 W/cm2, up to 4 min and immediately tested for cell viability using four different parameters: vital staining for the determination of the rate of cell growth; [3H]-thymidine and [3H]-leucine incorporation as an indication of the rate of DNA and protein synthesis respectively; and cloning efficiency as a measurement of the cell ability to multiply. Two human normal cell lines used in our studies, FS11 foreskin fibroblasts and Wish cells, were relatively resistant to ultrasonic irradiation effect although the growth rate of the latter was somewhat affected, particularly after 2 or 4 min of irradiation. However, cells derived from either malignant melanoma or breast carcinoma were highly sensitive to irradiation as demonstrated by a reduction of 96% and 65%, respectively, in cloning efficiency even after irradiation for 1 min. A third tumor cell line derived from lung carcinoma was more resistant. Two normal clones derived from NIH/3T3 mouse fibroblasts were used. These clones revealed some degree of sensitivity, particularly after 4 min of irradiation. However, their murine-sarcoma-virus transformed counterparts were found to be even more sensitive at identical times of ultrasonic irradiation, although the differences are not as striking as demonstrated with cells from human origin.(ABSTRACT TRUNCATED AT 250 WORDS)

Persistent measles virus infection enhances major histocompatibility complex class I expression and immunogenicity of murine neuroblastoma cells

The effect of persistent measles virus infection on the expression of major histocompatibility complex (MHC) class I antigens was studied. Mouse neuroblastoma cells C1300, clone NS20Y, were persistently infected with the Edmonston strain of measles virus. The persistently infected cell line, NS20Y/MS, expressed augmented levels of both H-2Kk and H-2Dd MHC class I glycoproteins. Activation of two interferon(IFN)-induced enzymes, known to be part of the IFN system: (2′–5′)oligoadenylate synthetase and double-stranded-RNA-activated protein kinase, was detected. Measles-virus-infected cells elicited cytotoxic T lymphocytes that recognized and lysed virus-infected and uninfected neuroblastoma cells in an H-2-restricted fashion. Furthermore, immunization of mice with persistently infected cells conferred resistance to tumor growth after challenge with the highly malignant NS20Y cells. The rationale for using measles virus for immunotherapy is that most patients develop lifelong immunity after recovery or vaccination from this infection. Patients developing cancer are likely to have memory cells. A secondary response induced by measles-virus-infected cells may therefore induce an efficient immune response against non-infected tumour cells.

Effect of interferon on mouse cells chronically infected with murine leukaemia virus: kinetic studies on virus production and virus RNA synthesis.

NIH/3T3 cells chronically infected with the Moloney strain of murine leukaemia virus were incubated with interferon (IF). There was no effect on virus production during the first 4 h, but thereafter an antiviral state gradually developed, reaching a maximum at about 12 h. When IF was removed, the antiviral state (expressed in terms of inhibition of release of virus) remained constant for 10 h, after which there was an abrupt return to the normal rate of virus release. Analysis of IF-treated cells showed that there was a three to fourfold increase in the amount of virus RNA in the nucleus at 48 h after IF addition, and still a slight increase at 72 h. There were no increases in the amounts of virus RNA in the cytoplasm during 72 h after the addition of IF. These results agree with the postulate that IF inhibits a late stage in the maturation of virus in chronically infected cells.

Effect of interferon on exogenous murine leukemia virus infection

Abstract When interferon (IF)-treated NIH/3T3 cells were exogenously infected with the Moloney strain of murine leukemia virus (M-MLV), no viral progeny release was detected as long as IF remained in the medium. This was evidently an inhibition of the virus replication and not a consequence of interfering with the establishment of the infection since, when IF was removed either before infection or later after infection, virus release gradually approached the rate of untreated control after a temporary delay. Furthermore, a direct examination revealed that IF treatment had no effect on the formation of infectious centers. IF treatment led to a reduced viral RNA synthesis. A similar inhibition of viral RNA synthesis was observed when the potent protein synthesis inhibitor cycloheximide (CH) was added early after infection. However, when added at a late stage, the drug had no effect on viral RNA synthesis. It appears, therefore, that IF-induced inhibition of viral RNA synthesis is not a feedback consequence of an inhibition of a later step but, rather, an inhibition of some early step. This conclusion was substantiated by the finding that when IF was eliminated from pretreated cultures up to 10 hr before infection, progeny release was still affected.

Effect of interferon on human cells releasing oncornaviruses: An assay for human interferon

Abstract Human RD-114 cells, chronically infected with a feline RNA tumor virus, were treated with different concentrations of human interferon in order to determine the kinetics of development of the antiviral state in human cells. The amount of virus released was monitored by reverse transcriptase (RT) activity in the culture medium. Our results demonstrated that 4–6 hr after the addition of 60 units/ml of interferon, a 70–80% inhibition in the amount of virus released is observed. The antiviral state induced in RD-114 cells appears to be rather stable since, even 24 hr after interferon removal, an inhibition of over 70% in virus release is observed in cells treated with 60 units/ml. Human interferon and mouse interferon showed species specificity when tested for their effect on virus release from RD-114 and mouse NIH/3T3 (murine leukemia virus) cells. Human interferon was titrated on RD-114 cells by either monitoring the RT activity in the culture medium or determining the reduction in the number of plaques formed on cell monolayers, after infection with vesicular stomatitis virus. Both assays were identical in their sensitivity. The use of the RT assay for detection of human fibroblast interferon during its purification by ion-exchange chromatography is illustrated.

Structural characterization of erythroid and megakaryocytic differentiation in Friend erythroleukemia cells.

OBJECTIVE The aim of this study was to examine the structural characterization of erythroid and megakaryocytic cell differentiation in Friend erythroleukemic cells using spectral imaging and electron microscopy. MATERIALS AND METHODS Two variants of Friend erythroleukemia cells were treated with hexamethylene bisacetamide (HMBA) to induce differentiation: 1) MEL, which exhibit the normal phenotype and are susceptible to differentiation; and 2) the resistant R1 cells. The cells were analyzed by spectral imaging along with transmission and scanning electron microscopy. The expression of cell cycle regulatory proteins was analyzed by Western blotting. RESULTS Spectral imaging of HMBA-treated MEL and R1 cells stained by May-Grünwald-Giemsa and subjected to spectral similarity mapping revealed five morphologic cell types: proerythroblast-like cells, normoblast-like cells, reticulocyte-like cells, megakaryocytes, and apoptotic cells. In MEL cells, both megakaryocytic differentiation characterized by nuclear lobes and erythroid differentiation characterized by accumulation of hemoglobin were detected; R1 cells were not committed to terminal differentiation. HMBA-induced cell cycle arrest at G(1) affected the expression of regulatory proteins in a similar manner in both types of cells. Expression of cyclin-dependent kinase 4 decreased and expression of p21(WAF1) increased. The level of the underphosphorylated form of phosphorylated retinoblastoma protein increased, inducing a decrease in the level of c-myc. In addition, we detected a decrease in the expression of the anti-apoptotic regulator, Bcl-2, and an increased expression of the pro-apoptotic regulator, Bax. CONCLUSIONS Spectral imaging provides new insight for the morphologic characterization of erythroid and megakaryocytic cell differentiation as well as apoptosis. Image analysis was well correlated to cell cycle arrest and the expression of regulatory proteins.

An effect of interferon on the uncoating of murine leukaemia virus not related to the antiviral state.

Adsorption of murine leukaemia virus (MLV) to NIH/3T3 cells, as determined by analysing its reverse transcriptase activity in the cell membrane, was found to be unaffected by interferon (IFN). Virus penetration and uncoating were followed by quantifying intracellular virions in terms of sedimentable reverse transcriptase activity in the cytoplasmic fraction. The penetrating virions were found to accumulate to a higher level in IFN-treated cells than in untreated controls. Intracellular virions were uncoated in untreated cells shortly after their penetration, whereas their uncoating was delayed in the IFN-treated cells for 2 to 3 h. Neither virus uncoating nor the effect of IFN on this process appeared to require new protein synthesis, since both were unaffected by cycloheximide (CH).

INHIBITION OF MALIGNANT CELL PROLIFERATION BY CULTURE MEDIA CONDITIONED BY CARDIAC OR SKELETAL MUSCLE

The present work is an attempt to explain the high resistance of muscles to cancer development. We used primary cultures of rat skeletal and cardiac muscle, and examined the effect of the supernatant of these cultures (conditioned medium; CM) on proliferation of cancer cells. The results demonstrated that CM inhibited the proliferation of several types of malignant cells by more than 50%, without a significant inhibition on normal cells. Cell cycle analysis revealed that CM increased the number of cells in S and G2 phases, suggesting a cytostatic effect of CM. For defining the biological properties of the factor(s) which are present in the CM, skeletal muscle cultures were grown in chemically defined medium (serum free medium). The concentrated sample was applied to a Sephadex G‐50 column and three fractions were obtained. Only one fraction showed inhibitory activity. Four protein bands were observed in this fraction, as revealed by SDS‐PAGE. We suggest that some, or all of these proteins are responsible for inhibition of tumor cell replication.

A possible requirement for protein synthesis early in the infectious cycle of the murine sarcoma-leukemia virus

Abstract Treatment of mouse 3T6 cells with cycloheximide early after infection with the Harvey strain of murine sarcoma-leukemia virus [H-MSV(MLV)l reversibly inhibited virus-specific RNA synthesis and infectious particle production. This effect was most pronounced when the drug was applied from 2 to 4 hr after infection. By hybridization to a labeled viral DNA probe, nearly half of the intracellular virus-specific RNA was shown to cosediment with cellular polyribosomes at 3 hr after infection. This RNA was derived from parental viral genomes since the same amount of viral RNA was detected in polyribosomes in the presence of arabinosyl cytosine which prevents viral DNA synthesis and the subsequent transcription of new viral RNA. The binding of parental viral RNA appeared to be specific since viral RNA was released from polyribosomes following treatment with EDTA. These findings suggest that the MSV(MLV) genome may function as messenger for the synthesis of one or more virus-specific proteins early after infection. The alternative possibility that cycloheximide inhibits some cellular function needed for viral reproduction is not excluded by these results.

Effect of interferon on the penetration of murine leukemia virus and the binding of its genome RNA to polyribosomes at the early stage of infection.

SummaryThe effect of interferon (IFN) on the adsorption, penetration and subsequent binding of the incoming genome RNA of Moloney murine leukemia virus (MLV) to polyribosomes, was studied in NIH/3T3 mouse fibroblasts. Virus adsorption was assayed by determining reverse transcriptase activity in the inoculating virus stock and in the cell membrane fraction before and after 45 minutes of infection. Both measurements suggested that IFN had no effect on virus adsorption. Virus penetration was determined by measuring the amount of viral RNA in the cell cytoplasm at 45 minutes after infection. This amount was remarkably lower in IFN-treated than in untreated cells. This reduction was not due to inhibition of a possible induction of endogenous viral genetic information by the penetrating virions, but was proved to be a direct effect of IFN on virus penetration, which was related to the IFN-induced antiviral state. The effect of IFN on binding of parental genome RNA to polyribosomes was then investigated by analysing Crt hybridization kinetics of polyribosomal viral RNA at different time intervals after infection. While in untreated control cells maximal binding occured at 3 hours postinfection, this maximal binding was observed in IFN-treated cells at 5 hours postinfection. The distribution of viral RNA molecules between sub-cytoplasmic fractions at 3 hours after infection was, in IFN-treated cells, significantly different from that observed in the untreated cells.