Contributions of differential p53 expression in the spontaneous immortalization of a chicken embryo fibroblast cell line

Background

The present study was carried out to determine whether the p53 pathway played a role in the spontaneous immortalization of the SC-2 chicken embryo fibroblast (CEF) cell line that has been in continuous culture for over three years.

Results

The SC-2 cell line emerged from an extended crisis period with a considerably slower growth rate than primary CEF cells. The phenotype of the SC-2 cells changed dramatically at about passage 80, appearing smaller than at earlier passages (e.g., passage 43) and possessing a small, compact morphology. This morphological change coincided with an increase in growth rate. Passage 43 SC-2 cells expressed undetectable levels of p53 mRNA, but by passage 95, the levels were elevated compared to primary passage 6 CEF cells and similar to levels in senescent CEF cells. However, the high level of p53 mRNA detected in passage 95 SC-2 cells did not correlate to functional protein activity. The expression levels of the p53-regulated p21^WAF1 gene were significantly decreased in all SC-2 passages that were analyzed. Examination of the Rb pathway revealed that E2F-1 and p15^INK4b expression fluctuated with increasing passages, with levels higher in passage 95 SC-2 cells compared to primary passage 6 CEF cells.

Conclusion

The present study suggests that altered expression of genes involved in the p53 and Rb pathways, specifically, p53 and p21^WAF1, may have contributed to the immortalization of the SC-2 CEF cell line.     

Characterization of a new human glioblastoma cell line that expresses mutant P53 and lacks activation of the PDGF pathway

Summary We have established and characterized a new glioblastoma cell line, termed GT9, from a biopsy sample of a female adult patient with glioblastoma multiforme. The line has now undergone over 60 passages and has been successfully cultured after cryopreservation. Immunofluorescence analyses with a panel of monoclonal antibodies were positive for glial fibrillary acidic protein and vimentin, and negative for neurofilament, galactocerebroside, and fibronectin, a pattern typical of glial cells. Based on a tetraploid, the composite karyotype of GT9 cells included the loss of chromosome 10, gain of chromosome 7, and the presence of double minute chromosomes, three of the most common karyotypic abnormalities in glioblastoma. Sequence analysis of p53 cDNA revealed a homozygous double mutation at codon 249 (commonly mutated in aflatoxin-associated hepatocellular carcinoma) and codon 250. Moreover, there was a complete absence of wild-type p53. However, unlike the majority of human glioblastomas previously described, the expression of platelet-derived growth factor-B (PDGF-B), a potent mitogenic autocrine factor, was low in GT9 cells. The expression and phosphorylation of c-Jun and Jun-B, downstream mediators of the PDGF pathway, were also low. Thus, deregulation of the PDGF pathway does not appear to be involved in the pathogenesis of the GT9 glioblastoma. Conversely, Jun-D, a negative regulator of cell growth, was also low. In addition, Phosphorylated Egr-1, a recently reported suppressor of PDGF-B/v-sis-transformed cells, was also low, suggesting that the lack of activation of the PDGF pathway was not due to these suppressive mechanisms. The circumstance of a weak or inactive PDGF-B autocrine mechanism in human glioblastoma paired with a homozygously altered p53 suggests that the loss of suppressor function of p53 may be a major contribution to the transformed phenotype of these cells.     

Ineffectiveness of the presence of H-ras/p53 combination of mutations in squamous cell carcinoma cells to induce a conversion of a nontumorigenic to a tumorigenic phenotype

Human tumor cells have properties in vitro or in surrogate hosts that are distinct from those of normal cells, such as immortality, anchorage independence, and tumor formation in nude mice. However, different cells from individual tumors may exhibit some, but not all of these features. In previous years, human tumor cell lines derived from different tumor and tissue types have been studied to determine those molecular changes that are associated with the in vitro properties listed above and with tumorigenicity in nude mice. In the present study, seven cell lines derived from human tumors were characterized for p53 and ras mutations that may occur in SCC tumor phenotypes and for tumor formation in nude mice. This investigation was designed to examine whether co-occurrence of mutated ras and p53 lead to a malignant stage in the progression process. None of the seven cell lines contained mutations in the recognized "hot spots" of the p53 tumor suppressor gene, but four had a nonsense/splice mutation in codon 126 and a mutation in codon 12 of the H-ras gene. The remaining three cell lines had p53 mutations in intron 5, in codon 193, and a missense mutation in codon 126, respectively. Four of seven cell lines were nontumorigenic; two of these cell lines contained a nonsense p53-126 mutation and mutated ras; one had a missense mutation at codon 126 but no mutated ras; the the fourth had only a p53 mutation at codon 193. Two of the nontumorigenic cell lines were converted to tumorigenicity after treatment with methyl methanesulfonate or N-methyl-N-nitro-N-nitrosoguanidine with no apparent additional mutations in either gene. Our analysis revealed that there was a high frequency of genetic diversity and mutations in both p53 and H-ras. There was also a lack of a causal relationship in the presence of mutations in p53 and the cells ability to exhibit a malignant potential in nude mice.     

Simultaneous cellular and humoral immune response against mutated p53 in a patient with lung cancer

We recently identified several Ags recognized by tumor-infiltrating B lymphocyte-derived Ab using SCID mice and a xenografted non-small cell lung cancer system. One of these identified Ags was mutated p53 with a point mutation resulting in the alteration of codon 158 from Arg to Leu. The aim of this study was to ascertain whether cellular immunity against mutated p53 exists in the same patient together with humoral immunity. Two different nona peptides (mutated p53(150) and p53(155) peptides), including a mutated amino acid derived from p53, were synthesized according to the binding motif of HLA class I of the established cancer cell line A904L from the patient. Mediastinal lymph node lymphocytes of the patient were stimulated weekly with the peptides. The mutated p53(155) peptide-stimulated lymphocytes showed specific cytotoxicity against both autologous EBV-transformed B cells pulsed with mutated p53(155) peptide and A904L. The mutated p53(155) peptide-specific CTL clone in an HLA-Cw*0702 restriction was established and analyzed for its TCR usage. Clonotypic PCR using CDR3-specific primers was applied to the tumor tissue containing the tumor-infiltrating lymphocytes. The specific amplification of PCR was found in the tumor tissue. These results demonstrated that not only B lymphocytes producing specific Ab against the p53 protein, but also CTL against mutated p53, expressed in autologous lung cancer cells exist in the tumor tissue. This approach may allow us to better understand the mechanisms of T and B cell immunity against the same tumor Ag in cancer patients.     

Potentiation of a tumor cell susceptibility to autologous CTL killing by restoration of wild-type p53 function

Inactivation of p53 has been implicated in many types of tumors particularly in non-small cell lung carcinoma, one of the most common cancers in which p53 mutation has been frequently identified. The aim of this study was to investigate the influence of p53 status on the regulation of tumor susceptibility to specific CTL-mediated cell death. For this purpose, we used a cytotoxic T lymphocyte clone, Heu127, able to lyse the human autologous lung carcinoma cell line, IGR-Heu, in a HLA-A2-restricted manner. Direct genomic DNA sequencing revealed that IGR-Heu expresses a mutated p53 at codon 132 of the exon 5 which results in the loss of p53 capacity to induce the expression of the p53-regulated gene product p21(waf/CIP1). Initial experiments demonstrated that IGR-Heu was resistant to Fas, TNF, and TRAIL apoptotic pathways. This correlated with the lack of p55 TNFRI, Fas, DR4, and DR5 expression. The effect of wild-type (wt) p53 restoration on the sensitization of IGR-Heu to autologous CTL clone lysis was investigated following infection of the tumor cell line with a recombinant adenovirus encoding the wt p53 (Adwtp53). We demonstrate that the restoration of wt p53 expression and function resulted in a significant potentiation of target cell susceptibility to CTL-mediated lysis. The wt p53-induced optimization of tumor cell killing by specific CTL involves at least in part Fas-mediated pathway via induction of CD95 expression by tumor cells but does not appear to interfere with granzyme B cytotoxic pathway.     

Phenotypic heterogeneity in osteogenesis imperfecta: the mildly affected mother of a proband with a lethal variant has the same mutation substituting cysteine for alpha 1-glycine 904 in a type I procollagen gene (COL1A1).

A proband with a lethal variant of osteogenesis imperfecta (OI) has been shown to have, in one allele in a gene for type I procollagen (COL1A1), a single base mutation that converted the codon for alpha 1-glycine 904 to a codon for cysteine. The mutation caused the synthesis of type I procollagen that was posttranslationally overmodified, secreted at a decreased rate, and had a decreased thermal stability. The results here demonstrate that the proband's mother had the same single base mutation as the proband. The mother had no fractures and no signs of OI except for short stature, slightly blue sclerae, and mild frontal bossing. As a child, however, she had the triangular facies frequently seen in many patients with OI. On repeated subculturing, the proband's fibroblasts grew more slowly than the mother's, but they continued to synthesize large amounts of the mutated procollagen in passages 7-14. In contrast, the mother's fibroblasts synthesized decreasing amounts of the mutated procollagen after passage 11. Also, the relative amount of the mutated allele in the mother's fibroblasts decreased with passage number. In addition, the ratio of the mutated allele to the normal allele in leukocyte DNA from the mother was half the value in fibroblast DNA from the proband. The simplest interpretation of the data is that the mother was mildly affected because she was a mosaic for the mutation that produced a lethal phenotype in one of her three children.     

P53 integrity in the genetically engineered mammalian cell lines AHH-1 and MCL-5

Recently, a C to T transition mutation in exon 8 of the p53 gene has been identified in a subculture of the genetically engineered human lymphoblastoid cell line AHH-1 and this mutation was proposed to cause a loss of function of the p53 suppressor protein and may limit the use of this cell culture in genotoxicology test assays. This led us to investigate early passage cultures of AHH-1 and its derivative MCL-5 to determine the distribution of the mutation. In order to characterise the presence of mutations at the p53 locus, exon 8 was analysed using restriction enzyme analysis and automated sequencing to locate possible changes of sequence. Mutations were identified at codon 282, and treatment with the Msp1 restriction enzyme led to incomplete digestion suggesting the presence of heterozygosity at the site which was confirmed by sequencing. Our results indicate that the p53 gene is heterozygous at the interface between the codons 281 and 282 in both AHH-1 and MCL-5. An Annexin V labeling study was carried out and both AHH-1 and MCL-5 cell lines were shown to undergo DNA damage induced cell death after a 1-h exposure to MNNG.     

Functional studies of a germ-line polymorphism at codon 47 within the p53 gene.

A rare germ-line polymorphism in codon 47 of the p53 gene replaces the wild-type proline (CCG) with a serine (TCG). Restriction analysis of 101 human samples revealed the frequency of the rare allele to be 0% (n = 69) in Caucasians and 4.7% (3/64, n = 32) among African-Americans. To investigate the consequence of this amino acid substitution, a cDNA construct (p53 mut47ser) containing the mutation was introduced into a lung adenocarcinoma cell line (Calu-6) that does not express p53. A growth suppression similar to that obtained after introduction of a wild-type p53 cDNA construct was observed, in contrast to the result obtained by introduction of p53 mut143ala. Furthermore, expression of neither p53 mut47ser nor wild-type p53 was tolerated by growing cells. In transient expression assays, both mut47ser and wild-type p53 activated the expression of a reporter gene linked to a p53 binding sequence (PG13-CAT) and inhibited the expression of the luciferase gene under the control of the Rous sarcoma virus promoter (RSVluc). In the same assay, mut143ala did not activate the expression of PG13-CAT and produced only a slight inhibitory effect on RSVluc. These findings indicate that the p53 variant with a serine at codon 47 should be considered as a rare germ-line polymorphism that does not alter the growth-suppression activity of p53.     

The over-expression of p53 H179Y residue mutation causes the increase of cyclin A1 and Cdk4 expression in HELF cells

Down-regulation of p53 expression has been found in a broad range of human cancers and cell proliferation disorders, indicating that p53 plays a key role in cell cycle regulation and tumor suppression. In our current study, we transfected human embryonic lung fibroblast (HELF) cells with pcDNA3-wild-type p53 (pcDNA3-wtp53) plasmid, or pcDNA3-H179Y-mutated p53 (pcDNA3-mtp53) plasmid that mimics the mutation found in some human lung tumors, and further studied the role of p53 in the regulation of cell proliferation. Over expression of wild-type p53 caused cell cycle arrest at G1 phase with reduced cell size, decreased expression of cyclin D3, cyclin E, Cdk2 and Cdk4, and increased expression of p21. In contrast, over expression of H179Y-mutant p53 promoted G1 to S phase transition with enlarged cell size and increased cyclin A1 and Cdk4 expression in HELF cells. These results indicate that mutation at the p53 H179Y residue up-regulates cyclin A1 and Cdk4 expression, and promotes HELF cell proliferation.     

Isolation and characterization of a novel bladder cancer cell line: Inhibition by epidermal growth factor

Summary A novel continuous cell line, designated BC3c, was established from a surgical biopsy of an invasive solid transitional cell carcinoma of the bladder derived from an 82-yr-old Caucasian female. BC3c cells were near-triploid bearing multiple structural and numerical chromosome anomalies. The epithelial origin of the cancer cells was indicated by the expression of cytokeratins 8 and 19 as well as by the absence of mesenchymal markers. Polymerase chain reaction-restriction-fragment length polymorphisms and single-strand conformation polymorphism mutation detection assays did not reveal any mutations in H-ras codon 12 and K-ras codons 12 and 13. In addition, no mutation in specific hot-spot codons of the p53 gene and no accumulation of the p53 protein were observed. BC3c cells grew rapidly in vitro, even in the absence of exogenous growth factors, because they were found to stimulate their growth in an autocrine manner. BC3c cells were found to express the epidermal growth factor-receptor (EGF-r) abundantly, but in contrast to other established bladder cancer cell lines, human recombinant epidermal growth factor inhibited the cells’ proliferation in vitro. These features render the newly established bladder cancer cell line BC3c a useful tool for further experimentation.     

Restoration of wild-type p53 in drug-resistant mouse breast cancer cells leads to differential gene expression, but is not sufficient to overcome the malignant phenotype

We established a breast cancer cell line from a fast growing mouse WAP-SVT/t breast tumor. Cells from this line, SVTneg2, switched off T-antigen expression, carry a missense mutation at the p53 codon 242 (mouse G242 corresponds to human hot spot mutation G245), are malignantly transformed, highly aneuploid and very insensitive to apoptotic stimuli. To examine the influence of wild-type p53 (wtp53) restoration on the behavior of the SVTneg2 cells, we transfected these cells with wtp53 and generated three permanent cell lines expressing wtp53. Interestingly, restoration of p53 had no influence on chemotherapy sensitivity and the transformation capacity of these breast cancer cells, but markedly changed the gene expression of wtp53-dependent genes after doxorubicin treatment. We postulate that restoration of p53 leads to massive changes in gene expression and to a reduced proliferation rate, but is not sufficient to overcome the malignant phenotype and the chemoresistance of SVTneg2.     

Establishment of a cell line (SH-4) from pleural effusion of a patient with melanoma.

A continuous tissue culture line (SH-4) derived from pleural effusion cells of a patient with metastatic melanoma is described. The cells became established after five passages lasting a period of 5 months of slow growth. Doubling time of the continuous culture was 20 hr in passage 51. The cell line is now in passage 60. The cells of all passages examined appear spindle-shaped or oligodendritic as seen by light microscopy. Pigment deposition in the cells is light and incomplete, but more marked than in cells of the original pleural effusion as shown by electron microscopy. Chromosome complement shows a single peak with a modal number of 51.     

Further studies with a cell immortalization assay to investigate the mutation signature of aristolochic acid in human p53 sequences

To test hypotheses on the origins of p53 mutations in human tumors, novel strategies are needed for generating mutation spectra experimentally. To this end we developed an assay employing Hupki (Human p53 knock-in) mouse embryonic fibroblasts (HUFs). Here we examine p53 mutations induced by aristolochic acid I (AAI)), the carcinogen probably responsible for Chinese herbal nephropathy. Six immortalized cultures (cell lines) from 18 HUF primary cultures exposed at passage 1 for 48 h to 50 microM AAI harbored p53 mutations in the human DNA binding domain sequence of the Hupki p53 tumor suppressor gene. The most frequently observed mutation was A to T transversion, corroborating our previous mutation study with AAI, and consistent with the presence of persistent AAI-adenine adducts found both in DNA of exposed patients and in DNA of AAI-exposed HUF cells. One of the mutations was identical in position (codon 139) and base change (A to T on the non-transcribed strand) to the single p53 mutation that has thus far been characterized in a urothelial tumor of a nephropathy patient with documented AAI exposure. Of the seven p53 mutations identified thus far in >60 HUF cell lines that immortalized spontaneously (no carcinogen treatment), none were A:T to T:A transversions. In addition, no A to T substitutions were identified among the previously reported set of 18 mutations in HUF cell lines derived from B(a)P treatment in which transversions at G:C base pairs predominated.     

Genetic and fitness changes accompanying adaptation of an arbovirus to vertebrate and invertebrate cells

The alternating host cycle and persistent vector infection may constrain the evolution of arboviruses. To test this hypothesis, eastern equine encephalitis virus was passaged in BHK or mosquito cells, as well as in alternating (both) host cell passages. High and low multiplicities were used to examine the effect of defective interfering particles. Clonal BHK and persistent mosquito cell infections were also evaluated. Fitness was measured with one-step growth curves and competition assays, and mutations were evaluated by nucleotide sequencing and RNA fingerprinting. All passages and assays were done at 32 degrees C to eliminate temperature as a selection factor. Viruses passaged in either cell type alone exhibited fitness declines in the bypassed cells, while high-multiplicity and clonal passages caused fitness declines in both types of cells. Bypassed cell fitness losses were mosquito and vertebrate specific and were not restricted to individual cell lines. Fitness increases occurred in the cell line used for single-host-adaptation passages and in both cells for alternately passaged viruses. Surprisingly, single-host-cell passage increased fitness in that cell type no more than alternating passages. However, single-host-cell adaptation resulted in more mutations than alternating cell passages. Mosquito cell adaptation invariably resulted in replacement of the stop codon in nsP3 with arginine or cysteine. In one case, BHK cell adaptation resulted in a 238-nucleotide deletion in the 3' untranslated region. Many nonsynonymous substitutions were shared among more than one BHK or mosquito cell passage series, suggesting positive Darwinian selection. Our results suggest that alternating host transmission cycles constrain the evolutionary rates of arboviruses but not their fitness for either host alone.     

Mutational analysis of the human p53 gene in malignant melanoma

Nine metastatic melanoma cell lines and two melanocyte cell lines were analyzed for point mutations in highly conserved regions of the p53 gene. No mutations were detected in the two melanocytic cell lines and in eight melanoma cell lines. However, a C----T transition at codon 248, resulting in a substitution of tryptophan for arginine, was found in one melanoma cell line. On immunohistochemical staining, only this cell line showed reactivity for mouse monoclonal antibody 1801, which is immunoreactive with human p53 protein. The original paraffin-embedded specimen from which this mutant cell line was established was obtained, and sequence analysis detected the identical mutation in the p53 gene as that seen in the derived cell line. This is the first report indicating point mutations in the p53 gene in malignant melanocytic tissues.     

Point mutation of the p53 gene resulting in splicing inhibition in small cell lung carcinoma

The p53 gene is functionally inactivated mostly by point mutations resulting in amino acid substitutions in a wide variety of human cancers. We found a novel mutation of the p53 gene in a small cell lung carcinoma cell line, Lu-143. One of the allelic p53 genes was lost accompanied by loss of heterozygosity for chromosome 17. In the remaining allelic p53 gene, there was a single-base substitution of G to T at position 1 within the splice donor site of intron 7, and the mutated intron was not spliced out during the mRNA maturation process. As a result of this mutation, larger sized p53 mRNA was expressed and no p53 specific protein was detected in this cell line. These results suggest that mutations causing splicing abnormalities are one of the molecular mechanisms for the p53 gene inactivation in human cancer.