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PubMed2004

Cloning of differentially expressed genes in skin fibroblasts from centenarians.

Normal human fibroblasts undergoing serial passaging have been extensively used to identify genes linked with aging. Most of the isolated genes relate to growth retardation signals and the failure of homeostasis that accompanies aging and senescence. In contrast, there is still limited knowledge regarding the nature of the genes that influence positively the rate of aging and longevity. Healthy centenarians represent the best example of successful aging and longevity. Studies using samples from these individuals have proved very valuable for identifying a variety of factors that contribute to successful aging. The aim of the current work was to take advantage of skin fibroblast cultures established from healthy donors including centenarians in order to clone differentially expressed genes in centenarians. First, we demonstrate that centenarian derived cultures follow the typical Hayflick curve and they enter senescence after serial passaging. Application of differential screening techniques in minimally passaged cultures of four control donors of different ages (18-80 years old) and four centenarians has resulted in the cloning of six differentially expressed genes in centenarians. Four of the cloned genes, namely adlican, KBL, EST 38 and EST 39, were over-expressed in centenarians, while VDUP1 and OCIF were down-regulated in the same samples. We have also compared the expression levels of two representative cloned genes in cultures of human embryonic and adult fibroblasts to establish potential links with replicative senescence. Interestingly, VDUP1 was found over-expressed in late passage cells, while EST 39 was down-regulated in the same cultures. Thus our work demonstrates that a combination of the use of both biopsies derived cells and classical in vitro cells passaging will facilitate the better understanding of the biology of aging and longevity.

Adult↗
PubMed2000

Interleukin-12 delivered by biodegradable microspheres promotes the antitumor activity of human peripheral blood lymphocytes in a human head and neck tumor xenograft/SCID mouse model.

BACKGROUND: The role of cytokines in tumor regression is now well established. The major limitation for the clinical use of cytokines is the lack of a simple and effective protocol for the local and sustained delivery of cytokines to the tumor milieu. This study reports suppression of human head and neck squamous cell carcinoma (HNSCC) by human peripheral blood lymphocytes (HuPBL) following local, sustained delivery of interleukin-12 (IL-12) to tumors with biodegradable microspheres in a human/SCID mouse chimeric model. Materials and Methods Nondisrupted biopsy pieces (120 mg) of primary HNSCC were implanted s.c. into severe combined immunodeficient (SCID) mice and were expanded by serial passage in mice. Tumors were then titrated with different doses of allogeneic HuPBL by coengraftment of tumor pieces and HuPBL into the subcutis of SCID mice to determine whether the HuPBL possessed antitumor activity (the SCID/Winn model). The lymphocyte subsets that were responsible for the suppression of tumor engraftment were identified by selective depletion of the CD4+, CD8+, and CD56+ cells from the HuPBL prior to engraftment into mice. Attempts were then made to augment the antitumor activity of the HuPBL either by repeated intralesional bolus injections of recombinant human IL-12 (0.5 microg x 10 doses) or with a single dose of IL-12-loaded microspheres ( approximately 1.65 microg IL-12/mg microspheres, 2 mg microspheres/mouse). RESULTS: Successful engraftment of HNSCC was observed in 12 of 19 different patient samples. Normal histological architecture of tumor was maintained up to four serial passages in the SCID mice. After the first tumor engraftment, but not in subsequent passages, human immunoglobulin produced by plasma cells present in the tumor infiltrating lymphocyte population was detected in the mouse sera. Allogeneic human PBL displayed antitumor cytotoxic activity in a cell dose-dependent fashion when coengrafted with the tumors passaged in SCID mice. Lymphocyte subset depletion studies established that tumor suppression was dependent on both the CD8+ T lymphocytes and the CD56+ natural killer cells. Treatment of tumors with a single intralesional injection of IL-12-loaded microspheres was highly effective, resulting in the complete suppression of tumor engraftment in 50% of the mice. In contrast, treatment of tumors with repeated bolus IL-12 injections suppressed tumor engraftment only transiently and did not result in complete tumor rejection in any of the mice. CONCLUSION: The coengraftment of HNSCC and allogeneic lymphocytes into SCID mice provides a viable model with which to evaluate immunotherapeutic strategies for human cancer. The use of biodegradable microspheres for local sustained delivery of cytokines to augment lymphocyte mediated antitumor immunity within the tumor microenvironment provides a safer and simpler alternative to current cytokine immunotherapy protocols.

Animals↗
PubMed1991

Common mechanism of retrovirus activation and transduction of c-mil and c-Rmil in chicken neuroretina cells infected with Rous-associated virus type 1.

We previously described the isolation of the IC10 retrovirus which transduced the v-Rmil oncogene, a new member of the mil/raf gene family. This virus was generated during serial passaging of Rous-associated virus type 1 (RAV-1) in chicken embryo neuroretina (NR) cells and was selected for its ability to induce proliferation of these nondividing cells. IC10 was isolated after six passages of culture supernatants but was not detected in proliferating NR cells during early virus passages. In this study, we molecularly cloned and sequenced another v-Rmil-containing provirus, designated IC11, from NR cells infected at the third virus passage of the same experiment. Both IC11 and IC10 transduced only the serine/threonine kinase domain of c-Rmil. Comparison of v-Rmil and c-Rmil sequences indicated that amino-terminal truncation is sufficient to activate the mitogenic properties of c-Rmil. IC11 and IC10 have identical 3' ends but differ by their 5' RAV-1-Rmil junctions. The 3' ends of both viruses were generated by recombination between Rmil and env genes, involving partial sequence identity. The 5' RAV-1-Rmil junction of IC11 was formed by a splicing process between the RAV-1 leader and a 37-bp c-Rmil exon located upstream of the kinase domain. NR cells infected with this virus synthesize a unique Rmil protein. IC10 contains most of the gag gene recombined with v-Rmil and encodes a gag-Rmil hybrid protein. Serial passaging of IC11 in NR cells led to the formation of a gag-Rmil-containing retrovirus. These results indicate that IC11 represents an early step in transduction and that this virus further recombined with RAV-1 to generate IC10. They confirm our previously proposed model for the multistep generation of v-mil-transducing retroviruses. Therefore, activation and transduction of c-mil and c-Rmil, in NR cells infected with RAV-1, result from a common mechanism.

Amino Acid Sequence↗
PubMedDate not supplied

Progression of human endometrial adenocarcinoma heterotransplanted into nude mice from hormone-sensitive to hormone resistant growth.

We have used a human tumor nude mouse model involving heterotransplantation and serial passage of an estrogen receptor (ER) positive, progesterone receptor (PgR) negative human endometrial adenocarcinoma. The effects of estradiol treatment on tumor growth, ER activation and PgR induction were investigated two and four years after heterotransplantation. In Experiment I, two years after initial heterotransplantation, tumor growth and proliferative rate showed a dose-related decrease, ER was activated by estradiol treatment (measured through an increased amount of ER bound with high affinity to nuclear element(s) (ERhs) and PgR was induced. Two years later (Experiment II), the amount of ER1s (ER measured in cytosolic fraction) as well as of ERhs was lower than at the beginning of Experiment I. ER could again be activated by estradiol treatment and PgR was also induced. However, in this experiment no effect either of tumor growth or of proliferative rate was observed during the estradiol treatment. Our study therefore indicates that an estrogen non-sensitive growth can develop during serial passages in intact, non-treated female nude mice, although the capacity for ER activation and PgR induction is maintained.

Adenocarcinoma↗
PubMed1973

Acquisition of sequences homologous to host DNA by closed circular simian virus 40 DNA. 3. Host sequences.

A preparation of serially passaged simian virus 40 (SV40) DNA, in which at least 66% of the molecules contain covalently linked cellular DNA sequences, was digested to completion with the Hemophilus influenzae restriction endonuclease. Polyacrylamide gel electrophoresis of the digest showed that the majority of the cleavage products migrated as nine classes of fragments, each class defined by a particular molecular weight. These classes of fragments differ in molecular weight from the fragments produced by the action of the same enzyme on plaque-purified virus DNA. Three classes of fragments were present in less than equimolar amounts relative to the original DNA. The remaining six classes of fragments each contain more than one fragment per original DNA molecule. DNA-DNA hybridization analysis (using the filter method) of the isolated cleavage products demonstrated the presence of highly reiterated cell DNA sequences in two of the nine classes of fragments. A third class of fragments hybridized with high efficiency only to serially passaged SV40 DNA; the level of hybridization to plaque-purified virus DNA was low and there was essentially no hybridization with cell DNA immobilized on filters. It is suggested that this class of fragments contains unique host sequences. It was estimated that at least 27% of the sequences in the substituted SV40 DNA molecules studied are host sequences. The majority of these are probably of the nonreiterated type.

Animals↗
PubMed1985

Altered patterns of keratin synthesis in human epidermal keratinocytes transformed by SV40.

Transformation of human epidermal keratinocytes by the oncogenic virus SV40 is a stage-specific process in which normal patterns of differentiation are progressively altered over time following infection. Within the context of this scheme, we examined the keratins produced by the infected cells. Immunofluorescence studies indicated that viral infection led to the formation of variant cells visibly lacking the normal keratin cytoskeleton after about 10-15 serial passages (60-90 cell generations) post infection. Analyses of variant cell formation in clonal populations grown on palladium islands revealed that the variants were derived within 2-3 cell divisions from cells containing an apparently normal keratin cytoskeleton, but that variant formation depended upon cell density. Immunoprecipitation of 35S-methionine labelled keratins from the infected keratinocytes revealed a gradual loss of the normal 46, 50, 56 and 58Kd keratin species over a period of many months after infection. The loss of the normal keratins was accompanied by the appearance of at least two species in the 48-52Kd size range not present in uninfected cells and the enhancement of a third, 40Kd, protein quite early after infection. Analysis of the altered keratin patterns on two-dimensional acrylamide gels using either isoelectric focusing (IEF) or non-equilibrium pH gradient electrophoresis (NEPHG) along the first dimension showed that the infected cells produced basic keratins which increased in relative abundance as cells became more transformed with serial passage including at least five isoelectric forms not seen in uninfected cells. Translation of poly A+ RNAs from the infected cells indicated that the altered keratin synthesis probably reflects changes in the translatable mRNA pool.

Cell Count↗
PubMed2004

Presence of an encephalomyocarditis virus internal ribosome entry site sequence in avian infectious bronchitis virus defective RNAs abolishes rescue by helper virus.

Avian infectious bronchitis virus (IBV) defective RNAs (D-RNAs) have been used for the expression of heterologous genes in a helper-virus-dependent expression system. The heterologous genes were expressed under the control of an IBV transcription-associated sequence (TAS) derived from gene 5 of IBV Beaudette. However, coronavirus D-RNA expression vectors display an inherent instability following serial passage with helper virus, resulting in the eventual loss of the heterologous genes. The use of the picornavirus encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) sequence to initiate gene translation was investigated as an alternative method to the coronavirus-mediated TAS-controlled heterologous gene expression system. IBV D-RNAs containing the chloramphenicol acetyltransferase (CAT) reporter gene, under EMCV IRES control, were assessed for IRES-mediated CAT protein translation. CAT protein was detected from T7-derived IBV D-RNA transcripts in a cell-free protein synthesis system and in situ in avian chick kidney (CK) cells following T7-derived D-RNA synthesis from a recombinant fowlpox virus expressing the bacteriophage T7 DNA-dependent RNA polymerase. However, CAT protein was not detected in CK cells from IRES-containing IBV D-RNAs, in which the IRES-CAT construct was inserted at two different positions within the D-RNA, in the presence of helper IBV. Northern blot analysis demonstrated that the IRES-containing D-RNAs were not rescued on serial passage with helper virus, indicating that the EMCV IRES sequence had a detrimental effect on IBV D-RNA rescue.

Animals↗
PubMed1995

Brain lesions in chickens experimentally infected with a neuroadapted strain of mesogenic Newcastle disease virus.

Neuroadapted Newcastle disease virus (Q10) was selected by tenth serial passage, in the chicken brain of a mesogenic strain (Q0) originally isolated from quails. Specific pathogen-free birds were inoculated intranasally with one of these viruses. At daily intervals for 7 days and then at 10, 14, and 21 days post-inoculation (PI), two birds from each group were killed and samples of the brain were collected for histopathological and virological examination. Q10 caused severe nonsuppurative encephalitis with nervous signs and high mortality. Lesions characterized by neuronal degeneration and necrosis, perivascular lymphocytic infiltration, and focal or diffuse astrogliosis occurred mainly in the parahippocampal cortex, hippocampus, hyperstriatum, neostriatum, subleptomeningeal and periventricular regions of the cerebrum. Spongy changes with neuronal degeneration and axonal spheroids were also observed in the brain stem of a few cases. The amount of virus in the brain reached a peak on day 4 PI and virus could not be recovered from the brain after 6 days PI. In contrast, Q0 caused nonfatal asymptomatic disease and virus could not be isolated from the brain, sections of which showed only minimal inflammatory changes. This difference in the lesions of the brain might be related to neurovirulence and, neuroadaptation by serial passage may occur by increased efficiency of viral replication in neurons.

Animals↗
PubMed1996

The equine herpesvirus 1 IR6 protein is nonessential for virus growth in vitro and modified by serial virus passage in cell culture.

The IR6 protein of different plaque isolates from three passages of the equine herpesvirus 1 strain Rac was investigated. Southern blot and DNA sequence analyses revealed that plaque isolates from the 12th passage (RacL11 and RacL22) retained both copies of the IR6 gene, whereas two different genotypes were observed by the 185th passage: RacM24 still harbored both copies of the IR6 gene, whereas RacM36 deleted one of the two copies. In the 256th passage (RacH), both copies of the IR6 gene were absent. As compared to the wild-type IR6 protein, both the RacM24 and RacM36 IR6 protein displayed amino acid exchanges at positions 34, 42, 110, and 134 of the 272 amino acid polypeptide. It is shown that (i) the IR6 protein is nonessential for virus growth in vitro. (ii) In RacL11-infected equine and rodent cells, the typical rod-like appearance of the IR6 protein could be detected from 6 hr p.i., whereas in RacM24- and RacM36-infected cells formation of these structures was not observed. (iii) The RacL11 IR6 gene product was present in both the nuclei and cytoplasmic fraction of infected cells. In contrast, the IR6 protein of both RacM24 and RacM36 colocalized with cytoplasmic membrane vesicles. (iv) The RacL11 and RacL22 IR6 protein is present in viral nucleocapsids, whereas that of RacM24 and RacM36 is not incorporated into virions. (v) The RacL11 IR6 gene product aggregated to disulfide-linked oligomers, whereas the RacM24 and RacM36 IR6 protein showed only marginal oligomerization. (vi) In COS7 cells transfected with constructs expressing either the full-length RacL11-IR6 protein or a truncated form lacking the 81 carboxyterminal amino acids, the formation of rod-like structures was observed, indicating that another viral protein is not necessary for aggregation of the IR6 protein. In contrast, the IR6 protein expressed from constructs derived from either RacM24 or RacM36 failed to form these structures. (vii) Analyses of chimeric RacL11-RacM24 IR6 proteins suggest a crucial role for amino acid Leu-134 in the ability of the IR6 protein to form the rod-like structures.

Amino Acid Sequence↗
PubMed1995

Immunomodulation of cultured vascular endothelial cells by serial cell passage.

UNLABELLED: Endothelial cell seeding has been successful in reducing the thrombogenecity of prosthetic vascular grafts in animal models, but results from clinical trials have been largely disappointing. These poor results have been associated with poor graft coverage in immediate seeding trials, and failure of cell culture in staged procedures. These problems could be largely overcome by utilising a bank of allogeneic endothelial cells, providing an ever ready supply. However, one potential pitfall with this technique would be the possibility of a rejection response following transplantation. AIM: To study the effects of prolonged tissue culture, on the ability of endothelial cells to generate an immune response. METHODS AND RESULTS: The immunogenecity of human umbilical vein endothelial cells was measured using the mixed lymphocyte endothelial reaction. It was demonstrated that prolonged tissue culture significantly reduced the immunogenecity of the cells, from a mean of 7261 cpm (S.E. +/- 243, n = 3) for cells of subculture 3, to 5478 cpm (+/- 156, p = 0.04) for cells of subculture 7 (p = 0.04, Wilcoxon paired rank test), but did not significantly impair morphology or antithrombotic function. CONCLUSION: This study provides evidence that prolonged tissue culture provides morphologically and functionally intact, immunomodified endothelial cells which may potentially be used in seeding prosthetic vascular grafts.

Blood Vessel Prosthesis↗
PubMed1974

Histochemical phosphatases and metachromasia in murine tumours induced by bone seeking radionuclides.

Tumours induced in mice, either CBA normal and chimaerical, or C3H, by (90)Sr or (226)Ra or plutonium have been examined histochemically with (1) diazotate fast red violet LB salt in naphthol AS-MX phosphate buffer at pH 8·6 and 5·2, (2) 1: 9 dimethyl methylene blue (Taylor).It is concluded:(a) The diagnosis of osteosarcoma is facilitated with Taylor's Blue which stains osteoid metachromatically. Cells of osteosarcoma, like normal osteoblasts, contain alkaline phosphatase but this may be lost by mutation either in the original tumour or subsequently on passage of the tumour serially to compatible hosts.(b) Osteosarcomata may contain giant-cells of two forms, bizarre tumour cells and osteoclasts; the latter contain acid phosphatase. Osteosarcomata which retain their osteoid on serial passage have few cells containing acid phosphatases.(c) Primitive mesenchymal cell tumours of angiomatous form may occur, if the bone marrow is irradiated, e.g. by (90)Sr-(90)Y and Pu. These tumours lack osteoid and cells interpretable as osteoblasts or osteoclasts (though they destroy bone).(d) Tumours classifiable as fibrosarcomata occur rarely, and may be truly of fibroblastic origin or be mutated osteosarcomata.(e) Lymphomata also occur when the marrow is irradiated ((90)Sr-(90)Y and Pu). They may be generalized, when their cells may contain alkaline phosphatase or lack it. They may be localized to abdominal viscera, the reticulo-sarcomatous form, in which case the cells lack alkaline phosphatase.

Acid Phosphatase↗
PubMed2000

Unusual distribution of mutations associated with serial bottleneck passages of human immunodeficiency virus type 1.

Repeated bottleneck passages result in fitness losses of RNA viruses. In the case of human immunodeficiency virus type 1 (HIV-1), decreases in fitness after a limited number of plaque-to-plaque transfers in MT-4 cells were very drastic. Here we report an analysis of entire genomic nucleotide sequences of four HIV-1 clones derived from the same HIV-1 isolate and their low-fitness progeny following 7 to 15 plaque-to-plaque passages. Clones accumulated 4 to 28 mutations per genome, with dominance of A --> G and G --> A transitions (57% of all mutations) and 49% nonsynonymous replacements. One clone-but not three sibling clones-showed an overabundance of G --> A transitions, evidencing the highly stochastic nature of some types of mutational bias. The distribution of mutations along the genome was very unusual in that mutation frequencies in gag were threefold higher than in env. Particularly striking was the complete absence of replacements in the V3 loop of gp120, confirmed with partial nucleotide sequences of additional HIV-1 clones subjected to repeated bottleneck passages. The analyses revealed several amino acid replacements that have not been previously recorded among natural HIV-1 isolates and illustrate how evolution of an RNA virus genome, with regard to constant and variable regions, can be profoundly modified by alterations in population dynamics.

Base Sequence↗