Therapeutic usefulness of wild-type p53 gene introduction in a p53-null anaplastic thyroid carcinoma cell line

Anaplastic thyroid carcinomas very often harbor the mutations in the tumor suppressor gene p53. We have previously shown that wild-type (wt) p53 gene introduction led to cell growth arrest, but not apoptosis, in p53-null anaplastic thyroid carcinoma cells. The present studies were designed to evaluate other therapeutic effects of wt-p53 gene introduction on p53-null thyroid carcinoma cells, as chemo- and radiosensitization and inhibition of angiogenesis have also been described recently as additional therapeutic advantages of wt-p53 gene introduction in tumor cells with p53 mutations. A p53-null anaplastic thyroid carcinoma cell line, FRO, and a FRO subline stably expressing a temperature-sensitive (ts) mutant of p53 (p53Val138), tsFRO, were used. ts-p53 functions as mutant and wt at nonpermissive (37 C) and permissive (32 C) temperatures, respectively. tsFRO showed a prolonged cell doubling time compared to parental FRO when cultured at 32 C, but the cell growth rate was similar between FRO and tsFRO at 37 C. The cytotoxic and clonogenic assays demonstrated that although the sensitivity to three different anticancer agents (cisplatin, 5-fluorocytosine, and doxorubicin) was unaltered, radiosensitivity was enhanced in tsFRO compared to FRO at 32 C. Unexpectedly, in studies on angiogenesis, expression levels of vascular endothelial growth factor (an angiogenic factor) messenger ribonucleic acid were similar between FRO and tsFRO, and thrombospondin-1 (an antiangiogenic factor) messenger ribonucleic acid and protein levels were about 2.5-fold lower in tsFRO than FRO at 32 C, although any difference could not be detected in their ability to inhibit in vitro angiogenesis with the culture medium conditioned by tsFRO and FRO at 32 C. These results suggest that p53-defective thyroid carcinomas may benefit from the combination of p53 gene therapy and radiotherapy. However, further study will be necessary to clarify the pathological significance of thrombospondin-1 in angiogenesis and thyroid tumor growth.     

Wild-type p53 and a p53 temperature-sensitive mutant suppress human soft tissue sarcoma by enhancing cell cycle control

Soft-tissue sarcomas are a heterogeneous group of tumors that are putatively mesenchymal in origin. Therapeutic advances in this disease have been limited over the past several decades. Approximately one-half of all patients will ultimately succumb, usually to uncontrollable pulmonary metastases. Although little is known about the underlying molecular determinants driving soft-tissue sarcoma inception, proliferation, and metastasis, mutation of the p53 gene is the most frequently detected molecular alteration in this disease. Accordingly, we were interested in determining whether transduction of wild-type (wt) p53 into soft-tissue sarcomas bearing mutated p53 genes might alter the malignant phenotype. SKLMS-1 is a human-derived leiomyosarcoma cell line with a codon 245 p53 point mutation. Cationic liposome was used to transfect wt p53 or 143Ala temperature-sensitive mutant p53 into this cell line. SKLMS-1 stable transfectants expressing wt p53 had decreased cell proliferation in vitro, decreased in vitro colony formation in soft agar, and decreased tumorigenicity in severe combined immunodeficient mice in vivo. Flow cytometric analysis of cell cycle components demonstrated markedly increased G1 cell cycle arrest and decreased entry into S phase, which corresponded to the induction of p21cip1 protein in the transfectants. Using SKLMS-1 stable transfectants expressing the 143Ala p53 temperature-sensitive mutant, we demonstrated the kinetics of and the causal relationship between wt p53 expression, the wt p53-dependent induction of cell cycle inhibitor p21cip1, and inhibition of cell cycle progression in p53-transfected SKLMS-1 cells. The ability to restore wt p53 growth-regulatory functions in soft-tissue sarcoma may ultimately be useful as a future therapy in patients with soft-tissue sarcomas.     

MYC abrogates p53-mediated cell cycle arrest in N-(phosphonacetyl)-L-aspartate-treated cells, permitting CAD gene amplification

Genomic instability, including the ability to undergo gene amplification, is a hallmark of neoplastic cells. Similar to normal cells, "nonpermissive" REF52 cells do not develop resistance to N-(phosphonacetyl)-L-aspartate (PALA), an inhibitor of the synthesis of pyrimidine nucleotides, through amplification of cad, the target gene, but instead undergo protective, long-term, p53-dependent cell cycle arrest. Expression of exogenous MYC prevents this arrest and allows REF52 cells to proceed to mitosis when pyrimidine nucleotides are limiting. This results in DNA breaks, leading to cell death and, rarely, to cad gene amplification and PALA resistance. Pretreatment of REF52 cells with a low concentration of PALA, which slows DNA replication but does not trigger cell cycle arrest, followed by exposure to a high, selective concentration of PALA, promotes the formation of PALA-resistant cells in which the physically linked cad and endogenous N-myc genes are coamplified. The activated expression of endogenous N-myc in these pretreated PALA-resistant cells allows them to bypass the p53-mediated arrest that is characteristic of untreated REF52 cells. Our data demonstrate that two distinct events are required to form PALA-resistant REF52 cells: amplification of cad, whose product overcomes the action of the drug, and increased expression of N-myc, whose product overcomes the PALA-induced cell cycle block. These paired events occur at a detectable frequency only when the genes are physically linked, as cad and N-myc are. In untreated REF52 cells overexpressing N-MYC, the level of p53 is significantly elevated but there is no induction of p21waf1 expression or growth arrest. However, after DNA is damaged, the activated p53 executes rapid apoptosis in these REF52/N-myc cells instead of the long-term protective arrest seen in REF52 cells. The predominantly cytoplasmic localization of stabilized p53 in REF52/N-myc cells suggests that cytoplasmic retention may help to inactivate the growth-suppressing function of p53.     

Studies of X-irradiated bladder cancer cell lines showing differences in p53 status: absence of a p53-dependent cell cycle checkpoint pathway

Cell growth arrest is a common response to DNA damage by ionising irradiation and the p53 gene has been shown to play an important role in this mechanism, possibly in a tissue-specific manner. Mutations in the p53 gene are frequent in invasive bladder cancers, which are often treated by radiotherapy. In this paper we have investigated the growth response to X-irradiation of three bladder cancer cell lines with differing p53 status: UCRU-BL-17 overexpresses mutant p53, while UCRU-BL-13 and UCRU-BL-28 contain wt P53. We have also examined the expression of proteins reported to be part of the p53 control pathway in response to irradiation-induced DNA damage. No G1 arrest was detectable in any of the cell lines after ionising irradiation; furthermore, in a downstream event reported to be correlated with p53 function there was no increase in WAF-1 protein levels regardless of p53 status. Rather, ionising irradiation resulted in G2 arrest, but the extent of this was not related to p53 status. p16 levels were also not affected by irradiation. Our results suggest that the UCRU-BL-28 cell line may have a defect in the p53-cell control pathway upstream of p53, while UCRU-BL-13 cells may have a defect downstream between p53 and WAF-1.     

Absence of p53-dependent cell cycle regulation in pluripotent mouse cell lines

We examined the expression of p53 in three lines of pluripotent embryonal carcinoma (EC) and ES cells. p53 mRNA and protein levels were constitutively high in two lines but absent from one. In the P19 line of EC cells neither p53 protein nor mRNA was detected. The first intron of the p53 gene in these cells had been invaded by a murine leukemia virus and there was extensive hypermethylation of the p53 gene accompanying its inactivation. In all three cell lines, irradiation resulted in arrest of the cells in the G2 but not in the G1 phase of the cell cycle despite the induction of p21cip1 in the cell lines expressing p53. Thus, the chromosomal stability of EC and ES cells appears to be not dependent on the p53 protein and we interpret our results to suggest that these cells may require the deletion of p53 dependent cell cycle regulation in order to become immortalized.     

Molecular events including p53 and k-ras alterations in the in vitro progression of a human colorectal adenoma cell line to an adenocarcinoma

The aim of the current study was to identify genetic abnormalities in human colorectal adenoma and carcinoma derived cell lines, and to determine whether the genetic changes which occur in vitro are relevant to the in vivo situation. Loss of 1p(33-35) region was shown to be the most common chromosome 1 abnormality and loss of heterozygosity (LOH) of the DCC gene and/or adjacent sequences was detected in all adenoma derived cells as well as the carcinoma cell lines. The level of p53 protein was also investigated as increased cellular p53 protein had previously been associated with mutation of the p53 gene. A further aim was to investigate genetic changes in our in vitro model of tumour progression, where the adenoma derived PC/AA cell line has previously been converted in vitro to two distinct tumorigenic phenotypes, producing either an adenocarcinoma or a mucinous carcinoma in athymic nude mice. Progression to the adenocarcinoma phenotype was shown to involve a specific chromosome 1 rearrangement, loss of both normal copies of chromosome 18 (although DCC gene sequences were retained), loss of the remaining wild type allele of k-ras resulting in homozygosity for the k-ras codon 12 mutation and increased cellular p53 protein as detected by SDS-PAGE Western blotting. The increase in p53 protein was shown not to be due to the acquisition of a mutation in the p53 gene. Interestingly, progression of the adenoma derived PC/AA cell line to the mucinous malignant phenotype did not involve any of these molecular rearrangements, suggesting that different genetically distinct pathways are involved in colorectal carcinogenesis. These studies show that the genetic changes in our in vitro model of human colorectal tumour progression are similar to those observed in in vivo studies.     

Relationships between p53 induction, cell cycle arrest and survival of normal human fibroblasts following DNA damage

It is now well established that in response to genotoxic stresses mammalian cells show an increased p53 protein levels and undergo cell cycle arrest at G1/S and G2/M checkpoints. But, the consequences of these cell cycle arrests on cell survival are not yet elucidated. In this study, we have analysed the relationships between p53 protein induction, cell cycle arrest and cell survival following exposure of normal human fibroblasts (NHFs) to various genotoxic agents such as cisplatin, UV radiation and gamma radiation. p53 protein accumulation and G2/M arrest arose at the same time following exposure to DNA damaging agents, suggesting that p53 is responsible for the G2/M block. However, following inhibition of p53 induction by an antisense oligonucleotide, this G2/M arrest is even more important and correlates with an enhanced sensitivity of NHFs to UV radiation. In addition, there appears to be a threshold in the response of NHFs to DNA damaging agents, p53 induction and cell cycle arrest being observed only with lethal UV doses. We show that: 1) there appears to be a threshold in the cellular response to genotoxic agents, below which neither p53 induction, nor cell cycle arrest, nor cell survival alteration occur and beyond which p53 induction is accompanied by cell cycle arrest and decreased cell survival; 2) although there is a tight temporal relationship, the onset of which depends of the DNA damaging agent used, between the start of p53 induction and the occurrence of G2/M arrest, this latter is independent of p53; 3) p53 inhibition enhances NHFs' sensitivity to DNA damaging agents, the extent of the G2/M arrest correlating with decreased cell survival. Finally, the lack of obligatory correlation between p53 inactivation, apoptosis and radio- or chemoresistance is discussed.     

p53 modulation of Fas/Apo-1 mediated apoptosis in a human renal cell carcinoma cell line

The mutant p53 gene was transfected into ACHN, a wild-type p53-containing human renal cell carcinoma (RCC) cell line. The colony forming efficiency in soft agar in the mutant-type p53-transfected cell line (ACHN/MP) was significantly higher than that in the vector-only transfected control cell line (ACHN/C). The anti-Fas monoclonal antibody (CH11) induced apoptosis in the ACHN/C cells in a dose-dependent manner, whereas the effect of CH11 on the ACHN/ MP cells was markedly suppressed. In addition, the cytotoxic effect of CH11 on the ACHN/MP cells was augmented by the pretreatment with interferon- , but the corresponding effect on ACHN/C cells was not. These findings suggest that Fas-mediated therapy could be a novel approach to RCC, if interferon- treatment is added according to the p53 gene status.     

Relationship of p53 overexpression to other cell cycle regulatory proteins in oral squamous cell carcinoma

Aberrations of the p53 gene and the overexpression of its protein are described in a variety of neoplasms, including oral and other head and neck cancers. Here we report the association of p53 (over)expression with a downstream cell cycle inhibitor p21/waf 1 in oral squamous cell carcinoma (SCC). The loss of expression of p16 and p27, two other cyclin-dependent kinase (cdk) inhibitors, was also examined. In this panel of tumours, 10/24 carcinomas were p53-immunopositive. Heterogeneous expression of p21 and p27 was seen in 10/24 SCC and 9/16 SCC, respectively, and this was not correlated to p53 status. The expression of p21 and p27 in these SCCs suggests the existence of mechanisms by which some growing tumour cells may tolerate these cell cycle inhibitors; eight SCCs lacked expression of both inhibitors but only two of these cancers overexpressed p53, suggesting that accumulation of p21/p27 can be independent of the functional status of the p53 gene. Data do not support a clear example of a phenotype that shows an overexpression of p53 with downregulation of p21 or p27 leading to cell cycle alterations. Furthermore, only three SCCs were p16-negative and p53-positive. This suggests that these two tumour suppressors may act in separate pathways.     

Epidermal growth factor-dependent cell cycle progression is altered in mammary epithelial cells that overexpress c-myc

Amplification and overexpression of the c-myc gene are common in primary human breast cancers and have been correlated with highly proliferative tumors. Components of the epidermal growth factor (EGF) receptor signaling pathway are also often overexpressed and/or activated in human breast tumors, and transgenic mouse models have demonstrated that c-myc and transforming growth factor alpha (a member of the EGF family) strongly synergize to induce mammary tumors. These bitransgenic mammary tumors exhibit a higher proliferation rate than do tumors arising in single transgenics. We, therefore, chose to investigate EGF-dependent cell cycle progression in mouse and human mammary epithelial cells with constitutive c-myc expression. In both species, c-myc overexpression decreased the doubling time of mammary epithelial cells by approximately 6 h, compared to parental lines. The faster growth rate was not due to increased sensitivity to EGF but rather to a shortening of the G1 phase of the cell cycle following EGF-induced proliferation. In cells with exogenous c-myc expression, retinoblastoma (Rb) was constitutively hyperphosphorylated, regardless of whether the cells were growth-arrested by EGF withdrawal or were traversing the cell cycle following EGF stimulation. In contrast, the parental cells exhibited a typical Rb phosphorylation shift during G1 progression in response to EGF. The abnormal phosphorylation status of Rb in c-myc-overexpressing cells was associated with premature activation of cdk2 kinase activity, reduced p27 expression, and early onset of cyclin E expression. These results provide one explanation for the strong tumorigenic synergism between deregulated c-myc expression and EGF receptor signal transduction in the mammary tissue of transgenic mice. In addition, they suggest a possible tumorigenic mechanism for c-myc deregulation in human breast cancer.     

p53 mutation and chemoresistance in oral-maxillofacial squamous cell carcinoma. Role of p53 in the cell cycle control and in the modulating action of chemotherapeutic agents

p53 is a "tumor suppressor gene" with a basic function in the cellular cycle control and subsequently in the induction of the neoplastic process. p53 found changed in the majority of malignant human tumors. In the oral and maxillofacial cancers p53 mutations varies from 4% to 60% of the cases. Researches in vitro in tumors of the colon-rectum, breast, lung, ovary, testicle, bladder and in leukaemia, show a correlation between p53 overexpression (mutation) and resistance to the anti-tumor agents. The functional connection between the p53 and the chemotherapic drugs action which cause a direct DNA damage, is the apoptosis, a physiologic mechanism activated by p53 for regulating cell growth but also indispensable for the cytotoxic effects (apoptosis is an irreversible process culminating in cell death). Loss of the p53 activity (mutation) in the tumoral cells determines non-activation of the apoptosis and the drug resistance. These results have led to early clinical applications. In the breast cancers the p53 is already utilized as chemoresistance marker, directing the therapy, if changed to alternative drugs (Taxolo) which have a p53-independent action. Even into cell lung cancers a retroviral vector containing the wild-type p53 gene was produced to mediate transfer of wild-type p53, noting tumor regression. In oral and maxillofacial tumors surgery is elective. It is emphasized that, in advanced cancers, it is included in multimode protocols where the neoadjuvant chemotherapy has an important clinical function, with precise indications. The possibility to determine previously the p53 "status" in the cancer cells by genetic study, may give a specific factor of screening, indicative of the tumor chemoresponse, together with other well-known prognostic factors, with advantage for the therapeutic programming and especially for the known surgical treatment.     

Penclomedine-induced DNA fragmentation and p53 accumulation correlate with reproductive cell death in colorectal carcinoma cells with altered p53 status

Acarbose reduces the absorption of monosaccharides derived from dietary carbohydrates, which play an important role in the metabolism and toxicity of some chemical compounds. We studied the effects of acarbose on the hepatotoxicity of carbon tetrachloride (CCl4) and acetaminophen (AP) in rats, both of which exert their toxic effects through bioactivation associated with cytochrome P450 2E1 (CYP2E1). Male Sprague-Dawley rats were kept on a daily ration (20 g) of powdered chow diet containing 0, 20, 40, or 80 mg/100 g of acarbose, with drinking water containing 0% or 10% of ethanol (vol/vol). Three weeks later, the rats were either killed for an in vitro metabolism study or challenged with 0.50 g/kg CCl4 orally or 0. 75 g/kg AP intraperitoneally. The ethanol increased the hepatic microsomal CYP2E1 level and the rate of dimethylnitrosamine (DMN) demethylation. The 40- or 80-mg/100 g acarbose diet, which alone increased the CYP2E1 level and the rate of DMN demethylation, augmented the enzyme induction by ethanol. The 40- or 80-mg/100 g acarbose diet alone potentiated CCl4 and AP hepatotoxicity, as evidenced by significantly increased levels of both alanine transaminase (ALT) and aspartate transaminase (AST) in the plasma of rats pretreated with acarbose. Ethanol alone also potentiated the toxicity of both chemicals. When the 40- or 80-mg/100 g acarbose diet was combined with ethanol, the ethanol-induced potentiation of CCl4 and AP hepatotoxicity was augmented. Our study demonstrated that high doses of acarbose, alone or in combination with ethanol, can potentiate CCl4 and AP hepatotoxicity in rats by inducing hepatic CYP2E1.     

Establishment and characterization of a multipotential neural cell line that can conditionally generate neurons, astrocytes, and oligodendrocytes in vitro

In the mammalian central nervous system (CNS), multipotential neural stem cells in the neuroepithelium generate the three major types of neural cells, namely, neurons, astrocytes, and oligodendrocytes. To explore the molecular mechanisms underlying proliferation and differentiation of these neural stem cells, we established a cell line named MNS-57 from the embryonic day 12 rat neuroepithelium by introducing the mycer fusion gene, in which c-myc can be conditionally activated by adding oestrogen to the culture medium. MNS-57 cells expressed nestin, vimentin, and the RC1 antigen, which are potential markers for neural stem cells. We show that under particular culture conditions, MNS-57 cells can conditionally generate neurons, astrocytes, and oligodendrocytes in vitro, indicating that they are likely to originate from multipotential neural stem cells. Incubating MNS-57 cells with either oestrogen, which activates mycer, or growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) stimulated their growth, and the combination of oestrogen and bFGF (or EGF) had a synergistically stronger mitogenic effect than the single factors. Furthermore, both c-myc activation and bFGF appeared to be necessary for the differentiation of MNS-57 cells, and only when stimulated by both signals simultaneously, the cells committed to generating multiple neural cell types. Thus, the property of the cell line is unique in that its differentiation into neurons and glia can be conditionally manipulated in vitro in an exogenous signal-dependent manner. We propose that the cell line described here will provide an useful in vitro model to understand genetic and environmental mechanisms that control the generation of neural cell diversity in the CNS.     

Geranylgeranylpyrophosphate, a metabolite of mevalonate, regulates the cell cycle progression and DNA synthesis in human lymphocytes

To determine the effect of coca chewing on heart rate (HR), mean arterial blood pressure (MAP), and plasma volume and their relationship with the hormones regulating cardiovascular and body fluid homeostasis, 16 male volunteers were examined at rest and during 1 h of cycle exercise at approximately 75% of their peak oxygen uptake in two trials separated by 1 mo. One trial was performed after the subjects chewed a sugar-free chewing gum (Coca- trial), whereas the other was done after the subjects chewed 15 g of coca leaves (Coca+), with the order of the Coca- and Coca+ trials being randomized. Blood samples were taken at rest, before (R1) and after 1-h chewing (R2), and during the 5th, 15th, 30th, and 60th min of exercise. They were analyzed for hematocrit, hemoglobin concentration, red blood cell count, plasma proteins, and for the fluid regulatory hormones, including plasma catecholamines [norepinephrine (NE) and epinephrine], renin, arginine vasopressin, and the atrial natriuretic peptide (ANP). During the control trial (Coca-), from R1 to R2, there was no significant change in hematologic, hormonal, and cardiovascular status except for a small increase in plasma NE. In contrast, it can be calculated that coca chewing at rest induced a significant hemoconcentration (-3.8 +/- 1. 3% in blood and -7.0 +/- 0.7% in plasma volume), increased NE and MAP, and reduced plasma ANP. Chewing coca before exercise reduced the body fluid shifts but enhanced HR response during exercise. These effects were not accompanied by changes in NE, epinephrine, renin, and arginine vasopressin plasma levels. In contrast, plasma ANP response to exercise was lower during the Coca+ trial, suggesting that central cardiac filling was reduced by coca use. It is likely that the reduction in body fluid volumes is a major contributing factor to the higher HR at any given time of exercise after coca chewing.     

Normalization of EGFR mRNA levels following restoration of wild-type p53 in a head and neck squamous cell carcinoma cell line

Hapalotrema mehrai Rao, 1976 and Hapalotrema postorchis Rao, 1976 (Digenea: Spirorchidae) are redescribed from the heart and pulmonary arteries of the green turtle, Chelonia mydas, from Moreton Bay in south-eastern Queensland. Hapalotrema pambanensis Gupta and Mehrotra, 1981 from C. mydas in India is made a synonym of H. mehrai. Hapalotrema dorsopora Dailey, Fast and Balazs, 1993 from C. mydas from Hawaii was described with a dorsally opening uterine pore, but this is found to be the opening of Laurer's canal; therefore H. dorsopora is also made a synonym of H. mehrai. In addition to differences in the numbers of testes and general dimensions, H. mehrai and H. postorchis differ in the development of Laurer's canal and in the absence of a canalicular seminal receptacle in H. postorchis.     

Proteolytic cleavage of p53: a model for the activation of p53 in response to DNA damage

p53 is a multifunctional protein that reacts to DNA damage within the cell and regulates the cell growth arrest and/ or apoptotic pathways. However, the mechanism of p53 activation in response to DNA damage is unknown. Recently we have shown that interaction of p53 with sites of DNA damage induces selective proteolytic cleavage of p53, resulting in fragments of 40 and 35 kDa molecular weight. We have also shown that interaction of p53 with single-stranded (ss)DNAs results in a different pattern of selective proteolysis. This interaction gives a novel of 50-kDa protein generated by C-terminal cleavage of the full length protein and released from the p53-ssDNA complexes. Here we discuss a model where p53 responds to the DNA damage by generating different sets of the proteolytic fragments according to the type of the damage.     

Gene mutations and increased levels of p53 protein in human squamous cell carcinomas and their cell lines

Using immunocytochemical and Western blotting techniques we have demonstrated the presence of abnormally high levels of p53 protein in 8/24 (33%) of human squamous cell carcinomas (SCC) and 9/18 (50%) of SCC cell lines. There was a correlation between the immunocytochemical results obtained with eight SCC samples and their corresponding cell lines. Direct sequencing of PCR-amplified, reverse transcribed, p53 mRNA confirmed the expression of point mutations in six of the positive cell lines and detected in-frame deletions in two others. We also detected two stop mutations and three out-of-frame deletions in five lines which did not express elevated levels of p53 protein. Several of the mutations found in SCC of the tongue (3/7) were in a region (codons 144-166) previously identified as being a p53 mutational hot spot in non-small cell lung tumours (Mitsudomi et al., 1992). In 11/13 cases only the mutant alleles were expressed suggesting loss or reduced expression of the wild type alleles in these cases. Six of the mutations were also detected in the SCCs from which the lines were derived, strongly suggesting that the mutations occurred, and were selected, in vivo. The 12th mutation GTG-->GGG (valine-->glycine) at codon 216 was expressed in line SCC-12 clone B along with an apparently normal p53 allele and is to our knowledge a novel mutation. Line BICR-19 also expressed a normal p53 allele in addition to one where exon 10 was deleted. Additionally 15 of the SCC lines (including all of those which did not show elevated p53 protein levels) were screened for the presence of human papillomavirus types 16 and 18 and were found to be negative. These results are discussed in relation to the pathogenesis of SCC and the immortalisation of human keratinocytes in vitro.