Re-expression of p16INK4a in mesothelioma cells results in cell cycle arrest, cell death, tumor suppression and tumor regression

Absence of expression of the p16IKN4a gene product is commonly observed in mesothelioma tumors and cell lines, while wild-type pRB expression is maintained. We have examined the biologic and potential therapeutic role of re-expressing p16INK4a gene product in mesothelioma cells and tumors. Following transduction with a p16INK4a expressing adenovirus (Adp16), over-expression of p16INK4a in mesothelioma cells resulted in cell cycle arrest, inhibition of pRB phosphorylation, diminished cell growth, and eventual death of the transduced cells. Expression of p16INK4a protein was accompanied by decreased expression of pRB as detected by immunoblot and immunohistochemistry. Experiments in mesothelioma xenografts demonstrated inhibition of tumor formation, tumor growth arrest and diminished tumor size and spread. p16INK4a gene product expression was also demonstrated in intraperitoneal xenografts of human mesothelioma cells. These results demonstrate that p16INK4a gene transfer may play a therapeutic role in the treatment of mesothelioma.     

Transrepression of lck gene expression by human T-cell leukemia virus type 1-encoded p40tax

To understand the mechanism of p56lck protein downregulation observed in human T cells infected by human T-cell leukemia virus type 1 (HTLV-1), we have investigated the ability of the 3' end of the HTLV-1 genome as well as that of the tax and rex genes to modulate p56lck protein expression and p56lck mRNA synthesis. By using Jurkat T cells stably transfected with constructs that expressed either the 3' end of the HTLV-1 genome (JK C11-pMTEX), the tax gene (JK52-Tax) or the rex gene (JK9-Rex), we found that the expression of p40tax (Tax) was sufficient to modulate p56lck protein expression. Similarly, we found that the expression of the mRNA which encoded p56lck was repressed in Jurkat T cells which expressed Tax. This downregulation was shown to be proportional to the amount of tax mRNA found in the transfected cells, as evidenced by experiments that used cells (JPX-9) stably transfected with a tax gene driven by a cadmium-inducible promoter. Furthermore, cadmium induction of Tax in JPX-9 cells transiently transfected with a construct containing the chloramphenicol acetyltransferase (CAT) gene under control of the lck distal promoter (lck DP-CAT) resulted in the downregulation of CAT gene expression. In contrast, cadmium induction of Tax in JPX-9 cells transiently transfected with a CAT construct driven by a lck DP with a deletion extending from position -259 to -253 (a sequence corresponding to a putative E-Box) did not modulate CAT gene expression, suggesting that the effect of Tax on p56lck is mediated through an E-Box binding protein.     

p16/INK4a and p15/INK4b gene methylation and absence of p16/INK4a mRNA and protein expression in Burkitt''s lymphoma

So-called sulfur-turf microbial mats, which are macroscopic white filaments or bundles consisting of large sausage-shaped bacteria and elemental sulfur particles, occur in sulfide-containing hot springs in Japan. However, no thermophiles from sulfur-turf mats have yet been isolated as cultivable strains. This study was undertaken to determine the phylogenetic positions of the sausage-shaped bacteria in sulfur-turf mats by direct cloning and sequencing of 16S rRNA genes amplified from the bulk DNAs of the mats. Common clones with 16S rDNA sequences with similarity levels of 94.8 to 99% were isolated from sulfur-turf mat samples from two geographically remote hot springs. Phylogenetic analysis showed that the phylotypes of the common clones formed a major cluster with members of the Aquifex-Hydrogenobacter complex, which represents the most deeply branching lineage of the domain bacteria. Furthermore, the bacteria of the sulfur-turf mat phylotypes formed a clade distinguishable from that of other members of the Aquifex-Hydrogenobacter complex at the order or subclass level. In situ hybridization with clone-specific probes for 16S rRNA revealed that the common phylotype of sulfur-turf mat bacteria is that of the predominant sausage-shaped bacteria.     

Characterization of p16(INK4A) expression in multiple myeloma and plasma cell leukemia

Loss of p16(INK4A) (p16) expression is frequently associated with the development of epithelial and lymphoid malignancies. However, the frequency and significance of p16 abnormalities in multiple myeloma (MM) and the more aggressive phase of plasma cell leukemia (PCL) have not been well defined. Accordingly, the goal of this study was to define the expression and function of p16 in fresh samples of MM and PCL. We found that p16 protein was highly expressed in primary MM cells, although it was undetectable in fresh samples of PCL. Additionally, p16 protein was also absent in four of four MM-derived cell lines. To determine the mechanism for p16 underexpression in PCL and MM-derived cell lines, we performed PCR analysis to evaluate both gene deletion and the presence of methylation. Interestingly, the p16 gene was present and methylated in all patient PCL cells and MM cell lines, whereas it was unmethylated in patient MM cells and normal B cells. Furthermore, treatment with the demethylating agent 5-deoxyazacytidine or p16 retrofection restored p16 protein expression and induced G1 growth arrest in patient PCL cells and MM cell lines. These results suggest that inactivation of the p16 gene by methylation may be associated with decreased growth control and the development of PCL in a subset of patients with MM.     

Trans-activator Tax of human T-cell leukemia virus type 1 enhances mutation frequency of the cellular genome

Two principles are presented that describe directional confounds associated with baseline differences: Principle 1: Change scores are confounded with baseline whenever data are skewed. Principle 2: When baseline differences are real, ANCOVA has a directional bias that magnifies differences in one direction and masks those in the other direction. Both principles involve a directional bias that is related to the direction of baseline difference and the direction of the hypothesized difference in change. Ethical dilemmas arise if decisions (whether or not to transform, whether or not to use ANCOVA) are chosen in order to maximize power by capitalizing on the directional bias and Type 1 error.     

Rb and p16INK4a expression in resected non-small cell lung tumors

Inactivation of the cyclin-dependent kinase inhibitor p16INK4a (CDKN2/MTS1) is documented in a wide variety of cancer cell lines and tumors. We have shown that loss of p16INK4a protein expression is a common event in early stage non-small cell lung cancer (NSCLC), correlates with a significantly worse survival, and is more common in higher stage disease. One hundred NSCLC tumors from patients undergoing definitive thoracotomies at a single institution were examined for p16INK4a and retinoblastoma protein (pRB) expression. Abnormal pRB staining was identified in 15% of the tumors, whereas 51% possessed aberrant p16INK4a protein expression. Tumors with aberrant expression of p16INK4a by immunohistochemistry were associated with a significantly worse survival (P=0.04). Additionally, the inverse correlation of pRB and p16INK4a expression previously noted in lung cancer cell lines and tumors was confirmed in this large cohort of patients, with 65% of the tumors demonstrating inverse expression of pRB and p16INK4a (p=0.00019). A statistically significant increase in aberrant p16INK4a expression, as well as inverse expression of p16INK4a and pRB, was seen with increasing pathological stage of disease. These findings establish the prognostic significance (of the absence of p16INK4, in resected NSCLC and confirm the critical importance of disrupting the pathway of cyclin-dependent kinase-mediated phosphorylation of pRB in the molecular oncogenesis and progression of NSCLC.     

Growth arrest and suppression of tumorigenicity of bladder-carcinoma cell lines induced by the P16/CDKN2 (p16INK4A, MTS1) gene and other loci on human chromosome 9

Wild-type P16/CDKN2 (p16INK4A, MTS1) cDNA, directed by the cytomegalovirus (CMV) immediate early promoter, was transfected into RT4 and RT112 bladder-carcinoma cell lines bearing a mutated endogenous P16/CDKN2 gene and lacking endogenous P16/CDKN2 respectively. In both cases, only transfected clones with rearranged exogenous P16/CDKN2 cDNA could be grown and propagated in cell culture. This result is reminiscent of transfection of wild-type p53 into cells with a deleted or mutated endogenous gene and suggests that P16/CDKN2, over-expressed under control of the strong CMV promoter, induces growth arrest in RT4 and RT112 cells. Transfer of human chromosome 9 to RT4 cells produced RT4/H9 hybrid clones retaining the P16/CDKN2 gene, since in RT4/H9 cell clones P16/CDKN2-gene expression is modulated by the physiological control of chromosomal regulatory sequence. All the RT4/H9 clones lost the entire chromosome 9, except clone 4 and clone 5, which maintained a deleted and an intact chromosome 9 respectively. Loss of several loci in 9p21, including P16/CDKN2, in tumors induced in nude mice by clone 4 and clone 5 suggests that P16/CDKN2 or other genes in 9p21 suppress tumorigenicity in bladder-carcinoma cells. Tumors induced by clone 4 and clone 5 show loss of markers in 9q. The regions 9q22.3, 9q32-33 and 9q34.2, which were maintained in the 2 clones and lost in their derived tumors, may contain tumor-suppressor genes relevant in bladder carcinoma. The results of this study suggest that the P16/CDKN2 gene controls growth of bladder-carcinoma cells when it is over-expressed, and may be involved in the development of bladder carcinoma, but other genes in 9p21 and 9q may participate in bladder-cancer progression.     

Transmission of human T-cell leukemia virus type 1 to mice

Human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia/lymphoma, HTLV-1-associated myelopathy/tropical spastic paraparesis, and other diseases. For prevention of the transmission of HTLV-1 and manifestation of these diseases, a small-animal model, especially a mouse model, would be useful. We injected HTLV-1-producing T cells (MT-2) intraperitoneally into neonatal C3H/HeJ mice. While the antibody against HTLV-1 antigens was not detectable in C3H/HeJ mice, HTLV-1 provirus was frequently detected in the spleen, lymph nodes, and thymus by PCR. HTLV-1 provirus was present at the level of 0 to 30 molecules in 10(5) spleen cells at the age of 15 weeks. In addition, a 59-bp flanking sequence of the HTLV-1 integration site was amplified from the spleen DNA by linker-mediated PCR and was confirmed to be derived from the mouse genome. HTLV-1 provirus was found in the T-cell fraction of the mouse spleen. These results indicate that mice can be infected by HTLV-1 and could serve as an animal model for the study of HTLV-1 infection and its pathogenesis in vivo.     

High-intensity Raf signal causes cell cycle arrest mediated by p21Cip1

Activated Raf has been linked to such opposing cellular responses as the induction of DNA synthesis and the inhibition of proliferation. However, it remains unclear how such a switch in signal specificity is regulated. We have addressed this question with a regulatable Raf-androgen receptor fusion protein in murine fibroblasts. We show that Raf can cause a G1-specific cell cycle arrest through induction of p21Cip1. This in turn leads to inhibition of cyclin D- and cyclin E-dependent kinases and an accumulation of hypophosphorylated Rb. Importantly, this behavior can be observed only in response to a strong Raf signal. In contrast, moderate Raf activity induces DNA synthesis and is sufficient to induce cyclin D expression. Therefore, Raf signal specificity can be determined by modulation of signal strength presumably through the induction of distinct protein expression patterns. Similar to induction of Raf, a strong induction of activated Ras via a tetracycline-dependent promoter also causes inhibition of proliferation and p21Cip1 induction at high expression levels. Thus, p21Cip1 plays a key role in determining cellular responses to Ras and Raf signalling. As predicted by this finding we show that Ras and loss of p21 cooperate to confer a proliferative advantage to mouse embryo fibroblasts.     

Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a

Oncogenic ras can transform most immortal rodent cells to a tumorigenic state. However, transformation of primary cells by ras requires either a cooperating oncogene or the inactivation of tumor suppressors such as p53 or p16. Here we show that expression of oncogenic ras in primary human or rodent cells results in a permanent G1 arrest. The arrest induced by ras is accompanied by accumulation of p53 and p16, and is phenotypically indistinguishable from cellular senescence. Inactivation of either p53 or p16 prevents ras-induced arrest in rodent cells, and E1A achieves a similar effect in human cells. These observations suggest that the onset of cellular senescence does not simply reflect the accumulation of cell divisions, but can be prematurely activated in response to an oncogenic stimulus. Negation of ras-induced senescence may be relevant during multistep tumorigenesis.     

Release of cell cycle constraints in mouse melanocytes by overexpressed mutant E2F1E132, but not by deletion of p16INK4A or p21WAF1/CIP1

Compared to normal melanocytes, melanoma cell lines exhibit overexpression of hyperphosphorylated retinoblastoma tumor suppressor protein (Rb) or a marked decrease in, or lack of, expression of Rb. Hyperphosphorylation of Rb results in increased E2F-mediated transactivation of target genes and cell cycle progression. Using a combination of gene disruption and ectopic expression in growth factor-dependent mouse melanocytes, we studied the roles of E2F1 and the p16INK4A and p21WAF1/CIP1 CKIs (cyclin dependent kinase inhibitors) in the acquisition of TPA (12-O-tetradecanoyl phorbol-13-acetate)-independent growth in culture, a hallmark of melanomas. Surprisingly, melanocytes from p16INK4A- or p21WAF1/CIP1-null mice remained TPA-dependent, and disruption of p21WAF1/CIP1 accelerated cell death in the absence of this mitogen. Disruption of E2F1 had the most profound effect on melanocyte growth, resulting in a fourfold decrease in growth rate in the presence of TPA. Furthermore, enforced overexpression of the DNA-binding-defective E2F1E132 mutant conferred TPA-independence upon melanocytes and was associated with sequestration of Rb and constitutive expression of E2F1 target genes, including p21WAF1/CIP1. We conclude that neutralization of Rb by E2F1E132, but not the disruption of p16INK4A or p21WAF1/CIP1, resulted in the accumulation of free E2F and cell cycle progression. Thus, mechanisms other than the loss of p16INK4A or p21WAF1/CIP1 that activate E2F may play an important role in melanomas.