Observations on cell lines derived from a patient with Hodgkin's disease.

Permanent cell lines have been established from a spleen nodule and lymph node of a male Hodgkin's disease (HD) patient whose father has the same disease. Th in vitro growth pattern morphological and cytogenetic characteristics of these lines maintained continuously for over 2 years are described. The cultures contain a population of mixed cell types that grow in suspension. Between 5 and 10% of the cells have surface immunoglobulins M and D. B-cell alloantigens are also detectable. While the cultures are predominantly lymphoid, some of the large cells, by light and electron microscopy, resemble the Reed-Sternberg and Hodgkin's cells of the original biopsies. Although the cells maintain the human diploid karyotype, they are heterotransplantable in nude mice. After 14 months of culture, chromosome rearrangement and losses, commonly seen in leukemic bone marrow, occurred. Close to 100% of the cells are Epstein-Barr nuclear antigen positive, but they lack Epstein-Barr viral (EBV) capsid antigen and EBV-induced early antigen. Nucleic acid hybridization tests indicated that there were no more than two EBV genome equivalents per cell. Tests with HD sera free of anti-EBV were negative. Electron microscope examination of the cells revealed the presence of intracellular as well as extracellular rare pleomorphic particles ranging from 400 to 1200 A. The nature of these particles, which increased in number after the cultures were treated with halogenated pyrimidines but not with dimethyl sulfoxide, remains questionable. The cultures derived from the mouse-passaged HD cells, however, had reverse transcriptase activity and readily identifiable type C particles which were probably of murine origin. These cultures have some unique features that make them useful in studying the perplexing pathological entity of HD.

Inverse polymerase chain reaction for cloning cellular sequences adjacent to integrated hepatitis B virus DNA in hepatocellular carcinomas.

Integration of hepatitis B virus (HBV) DNA is found in most HBV-related human hepatocellular carcinomas (HCCs). In the past, construction of genomic libraries was mainly employed to study the role of viral integration. However, large amounts of tissue DNA and a laborious screening procedures were required. Inverse polymerase chain reaction (IPCR) is based on the simple procedures of digestion of DNA with restriction enzymes and circularization of cleavage products before amplification using primers synthesized in the opposite orientations to those normally employed for PCR. This technique allows the in vitro amplification of DNA flanking a region of known sequence. By employing this method, starting from nanograms of hepatoma DNA, two adjacent cellular sequences were cloned from 11 HBV integrants in three HCCs. The original configurations in the chromosomes were further confirmed. One of the flanking cellular sequences was identified as the human 28S rRNA gene, the other was not found homologous to any known human sequences. This method appears to be practical and can be improved further to clone more flanking cellular sequences, especially in early and small HCCs.

Purification of RNA from animal cells using diethyl-pyrocarbonate.

Extraction of RNA from animal cells by a method using diethyl-pyrocarbonate yielded 50-60% of the total RNA. RNA purified by a hot phenol-SDS method from adenovirus 2 infected cells showed about 9% homology with adenovirus DNA, and RNA purified by diethyl-pyrocarbonate-SDS showed over 7% hybridization. Profiles of RNA prepared by both methods were identical when studied by polyacrylamide gel electrophoresis.

The mechanism of viral carcinogenesis by DNA mammalian viruses: RNA transcripts containing viral and highly reiterated cellular base sequences in adenovirus-transformed cells (DNA-RNA hybridization-viral-cell mRNA).

Virus-specific RNA was isolated from cells transformed by human adenovirus 2 and 7 by multiple hybridizations with and elutions from homologous viral DNA; RNA molecules purified by this selection procedure hybridized efficiently with both viral DNA (24-50%) and DNA from untransformed cells (12-27%). Virus-specific RNA isolated in the same manner from cells productively infected with adenoviruses did not hybridize significantly with cellular DNA. These findings suggest that RNA molecules containing covalently-linked viral and cellular sequences are transcribed in cells transformed by human adenoviruses. The high efficiency of hybridization with DNA from untransformed cells implies that viral DNA is integrated adjacent to highly reiterated cellular DNA sequences.

Variations in properties of virus released from morphologically different cell lines transformed in vitro by Friend leukemia virus.

The properties of the virus synthesized by each of three morphologically different cell lines originating from DBA/2J fetal liver cells transformed by the anemic strain of Friend leukemia virus in vitro were analyzed. The cells of line G-1 are malignant in syngeneic DBA/2 mice, grow in suspension, and are erythroid in origin. Cells of lines G-2 and G-3 are adherent, are epithelial in appearance, and produce no tumors in DBA/2J mice. Higher reverse transcriptase activity was detected in the culture fluid of lines G-2 and G-3, although virus from G-1 cells was more leukemogenic. Differences were also found in the virion density and size of the viral genome. RNA from the virions produced by G-2 and G-3 cells sedimented at 75 S in a sucrose gradient; virion RNA from G-1 cells sedmiented at 60 S. However, when subjected to polyacrylamide gel electrophoresis, all three virus strains showed identical RNA subunits with an estimated molecular weight of 2.6 X 10(6). Analysis of virion proteins by slab gel electrophoresis showed differences in envelope protein (gp71) components but not in the major core protein (p30). The properties of these viruses are stable and remain unchanged after passage in 3T3 cells.

Virus production and hemoglobin synthesis in variant lines of dimethyl sulfoxide-treated Friend erythroleukemia cells.

Variant Friend erythroleukemia cell clones were compared in regard to their response to dimethyl sulfoxide and in their abilities to synthesize virus and hemoglobin. Clear evidence was obtained that cellular growth is required for virus production. The effects of dimethyl sulfoxide on virus production were not observed in cell lines that were resistant to growth perturbation by the compound. Studies of cell variants that were defective in either hemoglobin or virus synthesis indicate that these activities are independently regulated.

Variations in the response of cloned murine friend erythroleukemia cells to different inducers.

Cells of the line 3BM-78 derived from murine bone marrow cells infected in vitro with polycythemic Friend leukemia virus (FLV-P) produce virus with spleen focus-forming activity (SFFV) and can be induced to synthesize hemoglobin. Fifteen clones, isolated from this line, have been analyzed in detail for the effect of different inducing agents (dimethyl-sulfoxide, DMSO; hexamethylene bisacetamide, HMBA; and sodium butyrate, SB) on the synthesis of hemoglobin and virus at the clonal level. All the clones proved to be inducible with one or more of the agents, but the degree of the response depended on the type and concentration of the agent used. In general, the effectiveness of the agent--within the usual range of concentration for induction--both for hemoglobin and for virus synthesis, was in the order HMBA greater than DMSO greater than SB. Reverse transcriptase activity was, however, more easily induced than hemoglobin synthesis in that stimulation was seen at lower concentrations of the same inducing agent. This clonal analysis confirmed that virus and hemoglobin production are regulated independently in these erythroleukemic cells chronically infected with FLV-P.

Transformation of DBA/2 mouse fetal liver cells infected in vitro by the anemic strain of Friend leukemia virus.

Fetal liver cells of DBA/2 mice were infected with the anemic strain of Friend leukemia virus (FLV-A), which has no spleen focus-forming virus (SFFV) activity. The infected cells were grown in medium with or without erythropoietin. Transformed lines were isolated only from the infected cultures that had been treated with erythropoietin at the time of their initiation. The properties of three permanent cell lines in serial passage for over 2 years are described. Each has an aneuploid karyotype. Only the immature hematopoietic cells of the first line have metacentric chromosomes. They grow in suspension, as do the erythroleukemic lines derived from leukemic spleens of FLV-infected mice, and clone on agar. They produce tumors resembling reticulum cell sarcomas upon subcutaneous inoculation into syngeneic hosts. Stimulation of differentiation induced after treatment with dimethyl sulfoxide identifies the cells of the first line as being erythroid in origin. The two other lines are adherent and epithelioid in appearance. These lines may have originated from the nonhematopoietic cells present in fetal liver. No tumors were produced after the subcutaneous inoculation of 10(6) cells. All three lines synthesize virus. The virus is attenuated for leukemogenicity and has no SFFV activity. The transforming event appears to be specific, because fetal liver cells from C57BL/6 mice, which are resistant to the induction of leukemia by FLV, were not affected by the virus. Malignant transformation of erythroid cells by FLV-A in vitro confirms the in vivo findings that SFFV may not be a necessary prerequisite for the induction of erythroleukemia in susceptible hosts.

Color flow mapping in ventricular septal defect: correlation of hemodynamic data and shunt flow area.

To evaluate the efficacy of two-dimensional Doppler echocardiography in assessing the severity of isolated ventricular septal defect, 31 children were studied within 24 hours of cardiac catheterization. The shunt flow area at peak systole (PSFA), diastole (DFA) and end-diastole (EDFA) and maximal shunt flow area (MSFA) were measured with frame-by-frame technique and corrected with body surface area and heart rate. The Qp/Qs ratio was calculated by the Fick's principle. Simultaneous pressures at peak systole (PSP), diastole (DP) and end-diastole (EDP) were recorded in the left (LV) and right ventricle (RV). The Qp/Qs ratio was correlated well with MFA (p = 0.05), MFA/BSA (p less than 0.001), PSFA/BSA (p less than 0.001), MFA/HR (p less than 0.05), MFA/BSA, HR (p less than 0.001), PSFA/BSA and HR (p,0.001). The PSP gradient between LV and RV was inversely correlated with MFA/BSA and PSFA/BSA (p less than 0.05) and HR (p less than 0.05). Two-dimensional Doppler echocardiography can provide a useful information of hemodynamic changes in children with isolated ventricular septal defect.

Inhibition of dimethyl sulfoxide-stimulated Friend cell erythrodifferentiation by hydrocortisone and other steroids.

Erythrodifferentiation and hemoglobin synthesis in dimethyl sulfoxide-stimulated Friend erythroleukemia cells were inhibited by hydrocortisone (HC) and four other steroids: dexamethasone, deoxycorticosterone, corticosterone, and aldosterone. The effect was specific, because no significant cytotoxicity occurred with any of these compounds at the concentrations that were inhibitory. The mechanism of action of HC was studied in detail. In the absence of dimethyl sulfoxide, it had no effect on hemoglobin levels; but, in the presence of this inducer, the synthesis of heme and globin were each inhibited by approximately 90%. There was no alteration in the synthesis of any major protein other than globin, as determined by gel electrophoresis of cell lysates. The activities of two enzymes in the heme biosynthetic pathway, delta-aminolevulinate dehydratase and uroporphyrinogen-I synthase, were inhibited by 80% and 70%, respectively. Globin mRNA induction was reduced by approximately 90%. This demonstrated that the HC inhibition of globin synthesis occurred at a pretranslational step. The dimethyl sulfoxide-induced single-stranded breaks in DNA, which have been suggested to play a role in Friend leukemia cell differentiation, were reduced in number but not eliminated. HC reduced the dimethyl sulfoxide-stimulation of virus release into the medium by approximately 50%. HC treatment in the absence of dimethyl sulfoxide doubled the production of virus.

Different hepatitis B virus core gene mutations in children with chronic infection and hepatocellular carcinoma.

BACKGROUND: The significance of mutations of hepatitis B virus (HBV) precore/core antigen in causing persistent infection and subsequent liver diseases is debatable. AIM: To investigate HBV core gene sequence changes in children with chronic HBV infection and their implications in hepatocellular carcinoma (HCC). METHODS: Thirty one chronic HBV infected children with documented hepatitis B e antigen seroconversion selected from 415 long term carrier children and 12 HBV related HCC children were studied. Four serial serum samples before and after hepatitis B e antigen seroconversion from each of the 31 children, and one serum sample taken from the 12 HCC children were subjected to HBV core gene sequence analysis. RESULTS: Mutations accumulated as chronic infection persisted and most frequently occurred at core gene codon 21 (29%), codon 147 (29%), codon 65 (16%), and precore stop codon 28 (74%) in the 31 chronic HBV infected children. Core gene mutation sites in HCC children were identified at core codons 74, 87, and 159. HCC children had more mutations in the core gene than those with chronic HBV infection (p=0.013). CONCLUSION: Accumulation of mutations of HBV core region in HCC children differ from those in chronic HBV infected children. This may be a clue to the pathogenesis of paediatric HCC.

Analysis of integrated hepatitis B virus DNA and flanking cellular sequences in a childhood hepatocellular carcinoma.

The DNA of tumor tissue K1 obtained at autopsy from a case of hepatocellular carcinoma (HCC) in a 9-year-old boy contained integrated hepatitis B virus (HBV) DNA at a single site in the chromosome (case 2, Chang et al.: Hepatology 13:316-320, 1991). To characterize further the integrated viral DNA sequences, a genomic library of the K1 DNA was constructed in the lambda L47.1 vector. One phage clone, designated KTM-1, containing integrated HBV DNA and cellular flanking sequences was obtained from this library. The restriction map and DNA sequence of this clone showed that the integrated HBV DNA was partially deleted and rearranged. The most conserved viral DNA sequences were surface and X genes and arranged in the opposite orientation. The viral core gene was not present. Using chloramphenicol acetyltransferase (CAT) assay, the C-terminal truncated X open reading frame was demonstrated to retain its trans-activating ability. The result suggested that the functional integrated X gene may play a role in hepatocarcinogenesis. The study also showed that the right cellular flanking sequences were human alphoid repetitive sequences.

High-dose intravenous gamma-globulin therapy in Kawasaki disease.

The efficacy for reduction of coronary aneurysm in Kawasaki disease was studied from 1984 to 1988 in Taiwan. One hundred and six children with Kawasaki disease were treated by one of the following regimens: regimen I: aspirin and 130-200 mg/kg/day of intravenous gamma-globulin (group I = 7), regimen II: 201-400 mg/kg/day of intravenous gamma-globulin with aspirin (group II = 49) and regimen III: aspirin alone (group III = 43) and no treatment (group IV = 7). By using two-dimensional echocardiography and aortography, the coronary arterial aneurysms noted in group I, II, III and IV were 42.9%, 49.0%, 44.2% and 16.7% respectively within 4 weeks of the illness and were 28.6%, 18.4%, 16.4% and 16.7% respectively during the follow-up period of 11.4 +/- 8.2 months. The incidence of coronary aneurysm was reduced significantly (p less than 0.005) in patients with high-dose gamma-globulin therapy and with aspirin therapy alone. However, there was no difference between group II and III, probably due to delays in the time of start of prophylactic gamma-globulin therapy. There was also significant lower incidence of the giant coronary aneurysm in children with high dose gamma-globulin therapy and with aspirin therapy. (p less than 0.05) The incidences of giant aneurysm in groups I, II, III and IV were 28.6%, 2.0%, 4.7% and 14.3% respectively. These results suggest that even with delay in the time of start of prophylactic gamma-globulin therapy, it still can reduce the formation of giant coronary aneurysm.

Low sodium content formula for infants with congestive heart failure.

Twenty-two infants of isolated ventricular septal defect with congestive heart failure were fed with lower-sodium content formula-Lonalac (Mead-Johnson) to study the clinical response of treatment for congestive heart failure. There were no significant changes of intake, urinary output, serum sodium, potassium and osmolality before, 2 days and 6 days after Lonalac feeding. The low sodium content formula may feed the infants with congestive heart failure in addition to the traditional anticongestive therapy.

Analysis of integrated hepatitis B virus DNA and flanking cellular sequences in the hepatocellular carcinoma cell line HCC36.

A hepatocellular carcinoma cell line, HCC36, was established from an adult HBV carrier in Taiwan. From Southern blot analysis, there were at least four sites of integration of HBV DNA, and no viral replicative intermediates were detected. A genomic library was constructed from HCC36 DNA, and two phage clones, designated lambda 36A and lambda 36B, were shown to contain HBV DNA and flanking cellular sequences. In lambda 36A, HBV DNA sequences were quite conserved, and 7.4% base variation was detected. The viral sequences in lambda 36A and lambda 36B differed in only four bases, in addition to the microdeletion and -insertion observed in lambda 36B. The flanking cellular sequences identified in lambda 36A were human Alu sequences and in lambda 36B satellite sequences.