Co-amplification of transcriptionally active epidermal growth factor receptor and ribosomal genes in the human hepatoma cell line Li7A.

A high level of expression of the functional product of the epidermal growth factor receptor (EGFR) gene was detected in the human hepatocarcinoma cell line Li7A and it was found to correlate with gene amplification. The karyotype was paratriploid, with 15 rearranged chromosomes, several of which contained abnormally banded regions (ABRs). The search for DNA sequences co-amplified with the EGFR gene, using the in-gel renaturation technique, allowed us to detect an amplified DNA band (La1) of about 30 kb. This DNA was used as a probe for in situ hybridization on chromosomes, to locate the amplified segment. In normal lymphocytes, the DNA of band La1 hybridized to chromosome regions in which repetitive DNAs are located, i.e. on juxtacentromeric regions, the site of alphoid and CCATT satellite DNA, and on the short arms of acrocentrics, the site of ribosomal RNA (RNR) genes. In Li7A cells, it hybridized to the same regions and, in addition, to several chromosome arms corresponding to ABRs. The same ABRs hybridized to EGFR and RNR probes, but neither Alu sequences nor various probes for other repetitive sequences were recognized. They also exhibited nucleolus organizer region staining characterizing functionally active (RNR) genes. It was concluded that transcriptionally active genes were co-amplified in the same ABRs, although they originated from different chromosomes, i.e. chromosome 7 for EGFR and acrocentrics for RNR genes.

DNA methylases separated through the HeLa cell cycle methodology show allosteric properties.

Two DNA methylases (DNAmets) can be separated through the cell cycle. The first appears as a minor peak in G1, the second as a major peak in S. Both enzymes protect from HpaII a plasmid (H31), constructed with the pBR322 vector (4.3 kbp) and the inverted A gamma fragment of the human globin gene (3.5 kbp), inserted at its HindIII site (the vector carries several HpaII sites, the insert only one HpaII site). DNAmets G1 and S show distinct Km values and different kinetics vs the ionic strength of the medium, while their Michaelis-Menten and Lineweaver-Burk plots are sigmoidal and hyperbolical curves, respectively. This is the first suggestion about the allosteric nature of the eukaryotic DNAmet system.

Amplification of T cells from human cord blood in serum-deprived culture stimulated with stem cell factor, interleukin-7 and interleukin-2.

We report the effects exerted by cytokine combinations, including stem cell factor (SCF), interleukin-7, interleukin-4 and interleukin-2, on the amplification of T cells from cord blood (CB) mononuclear cells cultured for 10-11 days under serum-deprived conditions. Of all the combinations investigated, SCF+interleukin-7 sustained the best fold increase (FI) of total nucleated cells (FI=6.4+/-1.17), amplifying preferentially CD4(+) over CD8(+) T-cell subsets (FI=4.72+/-0.79 vs 2.73+/-1.2, respectively, P<0.05). The addition of interleukin-2 to this combination did not significantly increase the total number of cells generated (FI=7.4+/-2.27), but allowed preferential amplification of CD8(+) over CD4(+) T cells (FI=6.04+/-0.14 vs 1.67+/-0.6, respectively, P<0.05). Single-strand conformation polymorphism analysis of the T-cell receptor V(beta)-chain rearrangements expressed by the expanded T cells indicated that the complexity of the T-cell repertoire had increased after 10 days of culture in the presence of SCF and IL-7. Interestingly, a modest expansion (FI=8.67+/-1.5) of myeloid progenitor cells was also observed in these cultures. These results indicate that it is possible to expand specific T-cell subsets for adoptive immunotherapy without losing myeloid progenitor cells necessary for neutrophil recovery after CB transplantation, by modulating the cytokines added to the cultures.

Characterization of the T cell receptor repertoire of neonatal T cells by RT-PCR and single strand conformation polymorphism analysis.

We have analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) the individual non-germ line configurations of the T cell receptor (TCR) Vbeta chains expressed by T cells from eight individual cord blood specimens. cDNA from each cord blood was amplified using a common primer coupled with a primer specific for each of 22 variable elements of the Vbeta chain family and the amplified fragments were separated under high resolution conditions. With cDNA from adult blood (as a control), all of the TCR chains were amplified as a smear consistent with the extensive polyclonality of adult T cells. In contrast, a heterogeneous pattern of amplification was observed with cDNAs from cord blood: only 26.7+/-21.9% of the 22 Vbeta chains analyzed were amplified as a smear. The majority of them were amplified as a discrete number of bands (up to 10) (in 68.2 +/-18.7% of samples) and some of them as a single fragment (4.0+/-7.8%). Only one of the eight samples analyzed expressed the majority (72.7%) of its Vbeta chains as a smear, consistent with an adult-like TCR repertoire. In conclusion, cord blood expressed, on average, a less complex TCR repertoire than adult blood.

Interleukin-6 production by human hepatoma lines is related to a low degree of cell differentiation.

In this study, we showed by Northern blot analysis and bioassays that scarcely differentiated human hepatoma cell line HA22T/VGH constitutively produces interleukin-6 (IL6). This cytokine was produced neither by moderately differentiated Li7A nor by well-differentiated HepG2 human hepatoma cell lines. The finding that transcripts for IL6 were not present in 2.2.15 cells, a HepG2-derived clone transfected with the intracellular replicative form of hepatitis B virus (HBV) DNA, suggests that production of this cytokine is not affected by HBV-encoded gene products, but is more likely related to the dedifferentiated state of hepatoma cells.

Human hepatoma cells expressing HLA class I molecules stimulate primary responses of purified CD8+ T lymphocytes.

In the present study, we investigated the ability of major histocompatibility complex (MHC) class-I+ human hepatoma cell lines to induce primary proliferative responses of purified allogeneic CD8+ T lymphocytes. We found that HA22T/VGH and Li7A, but not HepG2 cells induced significant proliferation of CD8+ T cells and that these responses were dependent on class I molecule expression. In blocking experiments carried out to identify the costimulatory signals involved, we found that anti-ICAM1 monoclonal antibodies drastically inhibited CD8+ T-cell proliferative responses. These findings suggest that transformed hepatocytes expressing HLA class I molecules may participate in anti-tumour immunosurveillance by the direct induction of cytotoxic T-cell responses through ICAM1-mediated adhesive interaction.

Heroin addicts infected by HBV and HIV have a low prevalence of HBV DNA in peripheral blood mononuclear cells.

Several reports show that the prevalence of HBV (hepatitis B virus) carriers in HIV (human immunodeficiency virus) infected populations is significantly higher than in HIV seronegative individuals, independent of the risk group for HIV, that is, homosexuals or drug abusers. In this context, evaluation of the simultaneous presence of HBV and HIV in PBMCs (peripheral blood mononuclear cells) is of particular interest for at least 2 reasons: 1) the possible reciprocal influence of the 2 viruses when they infect the same cell; 2) the possibility that HIV-induced hematological disorders could indirectly influence the settling of HBV in blood cell populations. We report data on the frequency of PCR positivity for HBV DNA in PBMCs from 62 HIV infected patients, rigorously selected by risk group, that is, intravenous use of heroin for at least 3 years and syringe promiscuity. Sixty-seven HIV negative individuals who never used any drug formed the control group. The analysis of the cases positive for HBV DNA in PBMCs showed that: 1) the overall prevalence of PCR positivity found in HIV infected patients was significantly lower than that registered in the control group; 2) PCR positivity among the subjects who were HBsAg negative and anti-HBV positive was extremely low in the HIV infected patients (3.7%) but quite frequent in the HIV negative subjects (37.0%). The results support the hypothesis that, unlike the HIV negative individuals, our HIV infected patients exhibited HBV DNA in PBMCS almost exclusively when they presented with active HBV replication.

Thymus-independent T-cell differentiation in vitro.

The generation of large quantities of novel human T-cell clones ex vivo would make a wide range of gene- and immuno-therapies for tumours and viral infections possible. Several techniques have been described to generate, in vitro and in vivo (using xenogenic hosts), mature T cells from fetal-neonatal and adult human CD34+ cells. All these techniques are cumbersome and cannot be easily translated into clinical protocols because they involve co-cultivation of CD34+ cells with thymic fragments from either human or murine fetuses. We report that the mononuclear cells of human cord blood contain a cell population that supports the differentiation of CD34+ cells into CD4+ or CD8+ naive T cells in serum-deprived cultures stimulated with stem cell factor and interleukin 7. CD4+ or CD8+ CD45RA+ TCRalphabeta+ T cells were continuously produced in vitro over a period of 20 d under these conditions. The generation of T cells in these cultures was a dynamic process and clones of T cells expressing new T-cell receptor beta-chain rearrangements were generated over time. These results pave the way for the development of very simple culture conditions for ex-vivo production of naive helper or cytotoxic T cells which could be very useful for gene- and immuno-therapy of human diseases.

Incidence of hepatitis B virus DNA and DNA-polymerase in sera of Italian asymptomatic carriers with the serological markers of HBV.

The presence of Hepatitis B virus (HBV) DNA in sera of patients with HBV related diseases is considered a reliable marker of virus active replication. In this paper HBV DNA was assayed by the molecular hybridization method with a 32P labeled nick translated HBV probe. The assay was positive in sera of 21 out of 22 HBeAg-positive and 4 out of 8 HBeAg-negative asymptomatic HBsAg carriers. 15 HBsAg-negative sera obtained from healthy donors showed no HBV DNA. Almost 80 per cent of HBeAg and HBV DNA positive sera revealed a DNA-polymerase activity. In order to determine the infectivity of HBsAg carriers, it appears that, whenever possible, the HBV DNA spot hybridization should be performed in conjunction with the DNA-polymerase, HBsAg and HBeAg tests.

Virus-free survival and down-regulation of CD4 in C8166 cells infected with human immunodeficiency virus type 1 at low density.

Compared with other T cell lines, C8166 lymphocytes are particularly susceptible to human immunodeficiency virus (HIV) infection and the outcome is invariably cell death. The results reported in this study demonstrate that the virus-induced cytolysis is strongly dependent on the initial cell density of C8166 cultures. Cultures diluted to 50 to 500 cells/ml almost completely maintained their cell duplication rate and released infectious virus into the medium. HIV infection of diluted C8166 cells is a simple and easily reproducible procedure for obtaining persistently infected cultures. These cultures contained genomic and extragenomic HIV DNA, the latter being assayed by PCR for two-long terminal repeat circular forms. The status of persistent infection disappeared within 2 months. The recovery is due to the replacement of CD4 down-regulated infected cells by overgrowing uninfected cell variants, which are transcriptionally inactive for CD4. The mechanisms underlying the emergence of these variants in persistently infected cultures are considered.

Human hepatoma cells expressing MHC antigens display accessory cell function: dependence on LFA-1/ICAM-1 interaction.

Malignant transformation of human hepatocytes is often accompanied by an increased expression of major histocompatibility complex (MHC) molecules, but whether this phenomenon is related to an enhanced immunogenicity remains unknown. In this study, we tested the capacity of a series of human hepatoma cell lines to induce proliferation of allogeneic T cells in primary mixed lymphocyte tumour cultures (MLTC). These cell lines were positive for class I molecules, whereas class II molecule expression was either constitutive or inducible by treatment with interferon-gamma (IFN-gamma). We found that HA22T/VGH cells expressing class II molecules constitutively stimulated high proliferative responses of purified CD4+ T lymphocytes, whereas class II-negative Li7A cells stimulated CD4+ T-cell responses only when induced by treatment with IFN-gamma. HA22T/VGH and Li7A cells also exerted a significant stimulatory activity for purified CD8+ T cells whereas HepG2 cells, in which MHC class II molecules are neither constitutive IFN-gamma-inducible, were unable to induce CD4+ and CD8+ T-cell proliferative responses. Phenotypical analysis revealed that HA22T/VGH and Li7A expressed high levels of intracellular adhesion molecule-1 (ICAM-1) and experiments with blocking monoclonal antibodies (mAb) demonstrated that this molecule played a key role in mediating the co-stimulatory function of hepatoma cells. In addition, HA22T/VGH cells were found to produce mRNA for interleukin-1 (IL-1) beta and IL-6, while Li7a only produced IL-1 beta, yet both these cytokines were found to play a small part, if any, in T-cell co-activation. On the whole, these results show tht hepatoma cells expression MHC antigens and ICAM-1 are able to deliver signals necessary for activation of resting CD4+ and CD8+ T cells and suggest that they may actively participate in the anti-tumour immune response.

Cell-cycle dependence and properties of the HeLa cell DNA polymerase system.

Analysis of the properties of the DNA polymerase (pol) system as a function of fundamental factors of the assay environment allowed a rather accurate estimation of its dependence on the HeLa cell cycle. For pol alpha, the temperature and pH optima were 38.1 degrees C and 8.0, respectively; for pol beta, these optima were 36.2 degrees C and pH 7.4. Pol gamma showed a pH optimum at 7.7. Optimum activity for both the alpha and beta enzymes was observed at 60 mM Tris. The maximal activity at 36.2 degrees C and pH 7.4 was associated with resistance to N-ethylmaleimide (MalNEt), whereas that at 38.1 degrees C and pH 8.0 was sensitive to MalNEt. Incorporation of [3H]dTTP was maximal after 1 hr of incubation for the former activity and after 4 hr, for the latter. In extracts from cells in early S phase, the pol activity decreased after 1 hr of incubation, was MalNEt-resistant, and was characterized by temperature and pH optima at 36.2 degrees C and 7.4, respectively. In extracts of late S-phase cells, the pol-catalyzed incorporation of [3H]dTTP continued after 4 hr of incubation, was MalNEt-sensitive, and was characterized by temperature and pH optima at 38.1 degrees C and 8.0, respectively. Thus, a pol beta-type activity appeared in early S phase, whereas a pol alpha-type activity appeared in late S. During the G1, M, and G2 phases, a background level of pol activity was observed that showed intermediate kinetic properties.

Trans-activation of epidermal growth factor receptor gene by the hepatitis B virus X-gene product.

The expression of the cellular gene coding for the epidermal growth factor (EGF) receptor (EGF-R) was assayed in the presence of hepatitis B virus (HBV) gene expression under different experimental conditions in human hepatoma-derived cells. First, transfection experiments of the well-differentiated HepG2 human hepatoma cell line using different expression vectors of the HBV X-region demonstrated that the X-gene product is capable of inducing EGF-R gene overexpression; in addition, by using a stable in vitro expression system for HBV, it was shown that EGF-R gene expression in these cells is greater than in the uninfected parent cells, and that this results in a three-fold increase in 125I-EGF binding. Finally, a CAT-expression assay was performed, indicating that regulatory regions of the EGF-R-gene are target sequences for X-protein trans-activation.

Accentuated response to phenylhydrazine and erythropoietin in mice genetically impaired for their GATA-1 expression (GATA-1(low) mice).

The response of mice genetically unable to up-regulate GATA-1 expression (GATA-1(low) mice) to acute (phenylhydrazine [PHZ]-induced anemia) and chronic (in vivo treatment for 5 days with 10 U erythropoietin [EPO] per mouse) erythroid stimuli was investigated. Adult GATA-1(low) mice are profoundly thrombocytopenic (platelet counts [x 10(9)/L] 82.0 +/- 28.0 vs 840 +/- 170.0 of their control littermates, P <.001) but have a normal hematocrit (Hct) (approximately.47 proportion of 1.0 [47%]). The spleens of these mutants are 2.5-fold larger than normal and contain 5-fold more megakaryocytic (4A5(+)), erythroid (TER-119(+)), and bipotent (erythroid/megakaryocytic, TER-119(+)/4A5(+)) precursor cells. Both the marrow and the spleen of these animals contain higher frequencies of burst-forming units-erythroid (BFU-E)- and colony-forming units-erythroid (CFU-E)-derived colonies (2-fold and 6-fold, respectively) than their normal littermates. The GATA-1(low) mice recover 2 days faster from the PHZ-induced anemia than their normal littermates (P <.01). In response to EPO, the Hct of the GATA-1(low) mice raised to.68 proportion of 1.0 (68%) vs the.55 proportion of 1.0 (55%) reached by the controls (P <.01). Both the GATA-1(low) and the normal mice respond to PHZ and EPO with similar (2- to 3-fold) increases in size and cellularity of the spleen (increases are limited mostly to cells, both progenitor and precursor, of the erythroid lineage). However, in spite of the similar relative cellular increases, the increases of all these cell populations are significantly higher, in absolute cell numbers, in the mutant than in the wild-type mice. In conclusion, the GATA-1(low) mutation increases the magnitude of the response to erythroid stimuli as a consequence of the expansion of the erythroid progenitor cells in their spleen.