A molecular blueprint for targeting cancer?
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Chromosome Aberrations↗
Biomedical subjects
T D Tlsty
Publications and source records attributed to T D Tlsty.
Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype.
Expression of the human telomerase catalytic component, hTERT, in normal human somatic cells can reconstitute telomerase activity and extend their replicative lifespan. We report here that at twice the normal number of population doublings, telomerase-expressing human skin fibroblasts (BJ-hTERT) and retinal pigment epithelial cells (RPE-hTERT) retain normal growth control in response to serum deprivation, high cell density, G1 or G2 phase blockers and spindle inhibitors. In addition, we observed no cell growth in soft agar and detected no tumour formation in vivo. Thus, we find that telomerase expression in normal cells does not appear to induce changes associated with a malignant phenotype.
Aspartic Acid↗
A novel coculture technique demonstrates that normal human prostatic fibroblasts contribute to tumor formation of LNCaP cells by retarding cell death.
The microenvironment influences the progression of an epithelial malignancy. To examine the effect of fibroblasts on epithelial cells by direct cell-cell contact in vitro, a coculture system was designed to assess cell death and proliferation of two cell populations when grown together. We used a green fluorescent dye to stain fibroblasts and distinguish them from unstained epithelial cells by a flow cytometer. We show that tumor cell death is 5-fold less when cocultured with normal human prostatic fibroblasts than when cultured alone. In contrast, proliferation of tumor cells was similar when cocultured with normal human prostatic fibroblasts or when grown alone. The reduction in tumor cell death during coculture appears to play a significant role in promoting tumor formation. Combination of prostatic fibroblasts with LNCaP xenografts formed large tumors at a high frequency with a low apoptotic index in vivo, whereas, LNCaP xenografts alone formed small infrequent tumors with a high apoptotic index. Therefore, prostatic fibroblasts promote tumor formation by retarding the apoptotic pathways in tumor cells.
Animals↗
An oncogenic form of p53 confers a dominant, gain-of-function phenotype that disrupts spindle checkpoint control.
Although it is well-established that p53 functions as a tumor suppressor gene, certain mutations exhibit gain-of-function activities that increase oncogenic transformation. We have found a common class of p53 missense mutation that exhibits a dominant, gain-of-function activity that generates genomic instability. Fibroblasts from Li-Fraumeni syndrome heterozygotes with such mutations generate polyploid cells when exposed to spindle depolymerizing agents. Expression of such mutant alleles in normal fibroblasts yields the same phenotype. This class of dominant, gain-of-function p53 mutation (p53(RSC), relaxed spindle checkpoint allele) does not require the transcriptional activation function of p53 for this behavior. Thus p53 mutations can contribute to progression of a cancer cell not only by absence of p53 tumor suppressor activity but also by the presence of an activity that promotes genetic instability.
Animals↗
Cell-adhesion-dependent influences on genomic instability and carcinogenesis.
Adhesion-dependent cell signaling is known to be important in carcinogenesis. It is postulated that several types of adhesion molecules act as tumor suppressor genes by enforcing cell-substrate and cell-cell adhesion thereby preventing the migration of cells and their invasion into surrounding tissues. Recent evidence, however, suggests that disruption of adhesion systems can both initiate neoplastic transformation and contribute a rate-limiting step to progression. Adhesion may modulate neoplastic processes by altering pathways that control genomic stability. Analysis of the adhesion-controlled inactivation of the p53 protein and the concomitant relaxation of cell cycle checkpoint control could identify the critical contributions of adhesion-mediated influences to carcinogenesis.
Cell Adhesion↗
Attenuation of G1 checkpoint function by the non-genotoxic carcinogen phenobarbital.
Non-genotoxic chemical carcinogens are capable of inducing tumors in rodents without interacting with or directly altering the genetic material. Since a preponderance of evidence suggests that cancer results from the accumulation of genetic alterations, the mechanisms by which many non-genotoxic carcinogens induce genotoxic events remain unclear. The present study investigated whether the mitogenic, non-genotoxic carcinogen phenobarbital (PB) could alter cell-cycle checkpoint controls, thereby indirectly leading to the accumulation of genetic damage. Initial studies involved characterizing cell-cycle checkpoint responses to DNA damage in freshly isolated B6C3F1 mouse hepatocytes. These cells responded to bleomycin-induced DNA damage by arresting in G1 and G2. Cell-cycle arrest was coupled with p53 protein induction; however, p21WAF1 protein levels remained unchanged. Studies that utilized hepatocytes isolated from C57BL p53-/- mice showed that the DNA damage-induced G1 cell-cycle arrest was dependent on p53 function, but cell-cycle arrest in G2 was not affected by loss of p53. PB was able to delay and attenuate the G1 checkpoint response without altering G2 checkpoint function. A reduction in p53 protein, but not transcript levels, was observed in hepatocytes exposed to PB. Additionally, PB delayed and attenuated p53 protein induction during DNA damage, which suggests that changes in the p53 protein may be contributing to the attenuated G1 checkpoint response caused by PB. Altered G1 checkpoint function represents an epigenetic mechanism by which phenobarbital may prevent the detection and repair of DNA damage and indirectly increase the frequency of genotoxic events above that occurring spontaneously. Abrogation of checkpoint controls may, thus, play an important mechanistic role in mitogenic, non-genotoxic chemical carcinogenesis.
Animals↗
Genetic and epigenetic influences in prostatic carcinogenesis (review).
Prostatic carcinogenesis is a multistep process with well-documented stages. Although prostate cancer is a major cause of mortality many small tumor foci never progress to form clinically significant disease, indicating that the disease process may be regulated at more than one level. Carcinogenesis is accompanied by increasing genetic damage to prostatic epithelial cells, however the pattern of genetic lesions is inconsistent. The differentiation of stromal cells surrounding tumors is more fibroblastic and less muscular than in normal prostate. The present communication reviews the roles of both genetic and, stromally derived, epigenetic effects on prostatic tumorigenesis.
Animals↗
Cellular adhesion regulates p53 protein levels in primary human keratinocytes.
To gain insight into p53 tissue-specific regulatory pathways and biological activities, we investigated mechanisms that may account for the elevated levels of p53 protein in human foreskin keratinocytes, relative to levels in dermal fibroblasts in vitro. Here, we report that the loss of cell anchorage resulted in an approximately 5-fold decrease in p53 levels in keratinocytes, which was reversible upon reattachment of cells to a substratum. In contrast, fibroblasts did not exhibit such adhesion-dependent regulation of p53 protein. Furthermore, p53 function was attenuated in keratinocytes relative to fibroblasts. These results link p53 to cell adhesion pathways and may provide a molecular basis for epigenetic differences in the maintenance of genomic stability among normal cell types.
Cell Adhesion↗
Genomic instability and its role in neoplasia.
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Animals↗
Identification of additional complementation groups that regulate genomic instability.
By somatic cell hybridization, amplification has been found to be a recessive genetic trait in three tumor cell lines examined. Studies with transgenic mice have shown that amplification frequency can be altered by a lack of wild-type TP53 (p53) activity. Other factors may regulate this phenotype in tumor cell lines possessing both wild-type p53 activity and amplification ability. Complementation analysis of somatic cell hybrids was performed to delineate groups of tumor cell lines that share a common defect that modulates the ability to amplify. The amplification frequencies of three normal fibroblast x tumor hybrids were suppressed 10-100-fold from parental tumor values, extending the observation that amplification is a recessive genetic characteristic in these cell lines. Analysis of tumor x tumor hybrids revealed at least two complementation groups. Defects in these groups differed from TP53 and implicate multiple variables in the regulation of gene amplification.
Antimetabolites, Antineoplastic↗
Separate pathways for p53 induction by ionizing radiation and N-(phosphonoacetyl)-L-aspartate.
The tumor suppressor gene product, p53, appears to be a significant participant in signaling pathways that mediate cellular responses to cytotoxic stresses. In particular, p53 appears to be a critical determinant of whether the cell lives or dies and how it progresses through the cell cycle after the cytotoxic exposure. Many of the molecular details for these signaling pathways remain to be elucidated, and whether all cytotoxic signals utilize the same pathway to increase p53 expression is not clear. Here, we demonstrate the existence of cell types in which the induction of p53 and associated G1 arrest by the antimetabolite, N-(phosphonoacetyl)-L-aspartate (PALA), is defective, whereas p53 induction and G1 arrest induced by ionizing radiation are intact. These observations demonstrate the existence of genetic defects that can alter p53 induction and associated cellular outcomes after some, but not all, cytotoxic insults and suggest distinct pathways of p53 induction by PALA and ionizing radiation.
Antimetabolites, Antineoplastic↗
Alteration of cell cycle kinase complexes in human papillomavirus E6- and E7-expressing fibroblasts precedes neoplastic transformation.
Expression of viral oncoproteins results in the loss of cell cycle checkpoint control and the accumulation of chromosomal abnormalities. Expression of both human papillomavirus type 16 oncoproteins, E6 and E7, in normal human fibroblasts completely dissociates p21 and proliferating cell nuclear antigen from the quarternary cyclin-cyclin-dependent kinase (CDK) complexes present in normal cells, causes disruption of the cyclin D-CDK4 complex and replacement with a CDK4-p16 complex, and leaves binary complexes of cyclin B1-CDC2 and cyclin A-CDK2 intact. These results are identical to those observed in fully transformed cells. The expression of the individual oncoproteins dramatically affects the association of proliferating cell nuclear antigen into the complexes while leaving the total cellular levels unaltered. Expression of low-risk human papillomavirus has no effect on cyclin complexes. These findings provide evidence for the gross alteration of cyclin-CDK complexes in preneoplastic cells and links this alteration to the loss of genomic stability.
Carrier Proteins↗
Regulation of genomic instability in preneoplastic cells.
Recent insights into the action of TP53 have uncovered signal transduction pathways that maintain genomic integrity. TP53 was the first gene demonstrated to be involved in these pathways, but mutation of several other genes can have a similar terminal effect. The characterization of these signal transduction pathways should provide further targets for the improvement of neoplastic diagnosis as well as therapeutic efficacy.
Cell Cycle↗
Defective G2 checkpoint function in cells from individuals with familial cancer syndromes.
The early events in the G2 checkpoint response to ionizing radiation (IR) were analyzed in diploid normal human fibroblasts (NHFs) and fibroblasts from patients with two heritable cancer syndromes. Exposure to gamma-radiation of asynchronously growing NHFs resulted in a rapid reduction in the number of cells in mitosis (G2 delay) and was accompanied by a quantitatively similar reduction in the p34CDC2/cyclin B in vitro histone H1 kinase activity as compared with sham-treated controls. This G2 delay was strong by 1 h following exposure to IR, maximal by 2 h, and was accompanied by an accumulation of tyrosine-phosphorylated p34CDC2 molecules. In contrast, fibroblasts from individuals with ataxia telangiectasia displayed significantly less reduction of the mitotic index or histone H1 kinase activity after IR. Low passage fibroblasts from individuals with Li-Fraumeni syndrome having one wild-type and one mutated p53 allele were similar to NHFs in their immediate G2 checkpoint response to IR, as were NHFs expressing the human papilloma virus type 16 E6 gene product (functionally inactivating p53) and low passage cells from p53-deficient mouse embryos. However, the p53-deficient fibroblasts were genomically unstable and became defective in their early G2 checkpoint response to IR. Furthermore, immortal Li-Fraumeni syndrome fibroblasts lacking wild-type p53 displayed an attenuated G2 checkpoint response. These results link the early events in G2 checkpoint response to IR in NHFs with a rapid inhibition of p34CDC2/cyclin B protein kinase activity and demonstrate that while not required for this immediate G2 delay, lack of p53 can lead to subsequent genetic alterations that result in defective G2 checkpoint function.
Ataxia Telangiectasia↗
Genomic instability and cancer.
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Animals↗
p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest.
gamma-Irradiation of human diploid fibroblasts in the G1 interval caused arrest of the cell cycle prior to S phase. This cell cycle block was correlated with a lack of activation of both cyclin E-Cyclin-dependent kinase 2 (Cdk2) and cyclin A-Cdk2 kinases and depended on wild-type p53. Although the accumulation of cyclin A was strongly inhibited in gamma-irradiated cells, cyclin E accumulated and bound Cdk2 at normal levels but remained in an inactive state. We found that both whole-cell lysates and inactive cyclin E-Cdk2 complexes prepared from irradiated cells contained an activity capable of inactivating cyclin E-Cdk2 complexes. The protein responsible for this activity was shown to be p21CIP1/WAF1, recently described as a p53-inducible Cdk inhibitor. Our data suggest a model in which ionizing radiation confers G1 arrest via the p53-mediated induction of a Cdk inhibitor protein.
Base Sequence↗
Differential disruption of genomic integrity and cell cycle regulation in normal human fibroblasts by the HPV oncoproteins.
Genomic integrity is maintained by a network of cellular activities that assess the status of the genome at a given point in time, provide signals to proceed with or halt cell cycle progression, and provide for repair of damaged DNA. Mutations in any part of these pathways can have the ultimate effect of disturbing chromosomal integrity. Recent work suggests that p53 performs this integrator function in mammalian cells. Our present study demonstrates that in mortal cells, the expression of E6 and E7 viral oncoproteins of type 16 human papillomavirus each disrupts the integration of these signals by diverged pathways. Cells expressing E6 protein, which binds and degrades the p53 protein, exhibited alterations in cell cycle control when placed in drug and displayed the ability to amplify the CAD gene. The expression of E7, which binds different cellular proteins important for transformation, including Rb, led to a p53-independent alteration in cell cycle control, a widespread cytocidal response, and polyploidy as a mechanism of drug resistance. These results demonstrate that diverse perturbations of molecular pathways can have different effects on chromosomal integrity.
Antineoplastic Agents↗
Clonal variation of tumorigenic potential in v-Ha-ras-transformed human bronchial epithelial cells: relationship to ras oncogene expression and CAD gene amplification.
Infection of an SV40 large-T antigen-"immortalized" human bronchial epithelial cell line with a Zip-v-Ha-ras retroviral vector resulted in a mass culture that was tumorigenic in athymic nude mice. A tumor cell line derived from passage of the mass culture in vivo, however, exhibited increased tumorigenicity and v-Ha-ras expression. To examine and compare the molecular events involving the ras oncogene during cell transformation in vitro and subsequent tumor formation in vivo, clonal cell populations were isolated from the v-Ha-ras-transformed mass culture. While the clonal cell lines exhibited diverse tumorigenic profiles, these differences did not correlate with v-Ha-ras expression. However, the expression of the activated ras gene, while not necessary for growth in vitro, did appear to be associated with a selective growth advantage in vivo. In addition, the modulation of gene amplification ability in these cells was not associated with the induction of tumorigenicity or v-Ha-ras expression.
Animals↗