Regulation Of Cell Cycle Control Research Articles

The ubiquitin ligase APCCdh1 is required to maintain genome integrity in primary human cells

Ensuring precise DNA replication and chromosome segregation is essential during cell division in order to provide genomic stability and avoid malignant growth. Proteolytic control of cell cycle regulators by the anaphase-promoting complex, activated by Cdh1 (APC(Cdh1)), is responsible for a stable G1 phase after mitotic exit allowing accurate preparation for DNA replication in the following S phase. APC(Cdh1) target proteins are frequently upregulated in tumor cells and the inactivation of human Cdh1 might interfere with genome integrity by target stabilization. Here we show that APC(Cdh1) is required for maintaining genomic integrity in primary human cells. Lentiviral-delivered strong and stable suppression of Cdh1 by RNA interference (RNAi) causes aberrant accumulation of several APC(Cdh1) target proteins, such as cyclin A, B, Aurora A or Plk1, which control accurate and equal distribution of the genetic information to daughter cells. This induces a premature and prolonged S phase, mitotic-entry delay and defects in chromosome separation and cytokinesis. Cell cycle deregulation by stable knockdown of Cdh1 leads to activation of p53/p21 and genomic instability, which is further increased by codepletion of p53. Thus, stabilization of APC(Cdh1) targets may initiate aberrant DNA replication and chromosome separation, and trigger a p53 response by deregulating G1 in primary human cells.

Nutrient Regulation of Tumor and Vascular Endothelial Cell Proliferation

Specific bioactive dietary components, such as the steroid receptor superfamily ligands vitamins A and D, have been studied extensively as potential cancer preventive and therapeutic agents due to their ability to regulate key processes in a variety of cell types which are dysregulated in neoplastic transformation namely, proliferation and differentiation. Alteration of one or more factors that regulate cell cycle control has been described as a predisposing event for early tumor development. In addition to tumor cell proliferation, the viability, growth and metastasis of solid tumors are also dependent on the vascularization of the tumor and establishment of blood flow. Both vitamins A and D exhibit anti-angiogenic properties which further strengthen their role as potential targets for the prevention and treatment of cancer. This review focuses on the role of vitamins A and D in preventing early tumor initiation and progression via control of the cell cycle in both tumor and vascular endothelial cells.

CDNA microarray analysis of cyclosporin A (CsA)-treated human peripheral blood mononuclear cells reveal modulation of genes associated with apoptosis, cell-cycle regulation and DNA repair

Cyclosporin A (CsA) is a potent immunosuppressant that has been extensively used to attenuate patient immune response following organ transplantation. The molecular biological mechanism of CsA has been extensively investigated in human T cells, and it has been shown to involve modulation of the intracellular calcineurin pathway. However, it is plausible that this chemical immunosuppressant certainly up- or down-regulate many other biochemical pathways of immune cells. In the present study, we used the cDNA microarray method to characterize the gene expression profile of human peripheral blood mononuclear cells (PBMC) treated in vitro with CsA and controls. The CsA treated PBMC displayed statistically significant induction of genes involved in the control of cell-cycle regulation (TRRAP), apoptosis/DNA repair (PRKDC, MAEA, TIA1), DNA metabolism/response to DNA damage stimulus (PRKDC, FEN1), transcription (NR4A2, THRA) and cell proliferation (FEN1, BIN1), whose data have permitted identification of target genes involved in CsA immunosuppression.

Identification of an effector protein and gain-of-function mutants that activate Pfmrk, a malarial cyclin-dependent protein kinase

Cyclin-dependent protein kinases (CDKs) are key regulators of cell cycle control. In humans, CDK7 performs dual roles as the CDK activating kinase (CAK) responsible for regulating numerous CDKs and as the RNA polymerase II carboxyl-terminal domain (CTD) kinase involved in the regulation of transcription. Binding of an effector protein, human MAT1, stimulates CDK7 kinase activity and influences substrate specificity. In Plasmodium falciparum, CDKs and their roles in regulating growth and development are poorly understood. In this study, we characterized the regulatory mechanisms of Pfmrk, a putative homolog of human CDK7. We identified an effector, PfMAT1, which stimulates Pfmrk kinase activity in a cyclin-dependent manner. The addition of PfMAT1 stimulated RNA polymerase II CTD phosphorylation and had no effect on the inability of Pfmrk to phosphorylate PfPK5, a putative CDK1 homolog, which suggests that Pfmrk may be a CTD kinase rather than a CAK. In an attempt to abrogate the requirement for PfMAT1 stimulation, we mutated amino acids within the active site of Pfmrk. We found that two independent mutants, S138K and F143L, yielded a 4–10-fold increase in Pfmrk activity. Significant kinase activity of these mutants was observed in the absence of either cyclin or PfMAT1. Finally, we observed autophosphorylation of Pfmrk that is unaffected by the addition of either cyclin or PfMAT1.

"Cullin 4 makes its mark on chromatin"

Cullin 4 (Cul4), a member of the evolutionally conserved cullin protein family, serves as a scaffold to assemble multisubunit ubiquitin E3 ligase complexes. Cul4 interacts with the Ring finger-containing protein ROC1 through its C-terminal cullin domain and with substrate recruiting subunit(s) through its N-terminus. Previous studies have demonstrated that Cul4 E3 ligase ubiquitylates key regulators in cell cycle control and mediates their degradation through the proteasomal pathway, thus contributing to genome stability. Recent studies from several groups have revealed that Cul4 E3 ligase can target histones for ubiquitylation, and importantly, ubiquitylation of histones may facilitate the cellular response to DNA damage. Therefore, histone ubiquitylation by Cul4 E3 ligase constitutes a novel mechanism through which Cul4 regulates chromatin function and maintains genomic integrity. We outline these studies and suggest that histone ubiquitylation might play important roles in Cul4-regualted chromatin function including the cellular response to DNA damage and heterochromatin gene silencing.

Perturbations of Cell Cycle Control in T Cells Contribute to the Different Outcomes of Simian Immunodeficiency Virus Infection in Rhesus Macaques and Sooty Mangabeys

In contrast to human immunodeficiency virus (HIV) infection of humans and experimental simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs), SIV infection of sooty mangabeys (SMs), a natural host African monkey species, is typically nonpathogenic and associated with preservation of CD4+ T-cell counts despite chronic high levels of viral replication. In previous studies, we have shown that the lack of SIV disease progression in SMs is related to lower levels of immune activation and bystander T-cell apoptosis compared to those of pathogenic HIV/SIV infection (G. Silvestri, D. Sodora, R. Koup, M. Paiardini, S. O'Neil, H. M. McClure, S. I. Staprans, and M. B. Feinberg, Immunity 18:441-452, 2003; G. Silvestri, A. Fedanov, S. Germon, N. Kozyr, W. J. Kaiser, D. A. Garber, H. M. McClure, M. B. Feinberg, and S. I. Staprans, J. Virol. 79:4043-4054, 2005). In HIV-infected patients, increased T-cell susceptibility to apoptosis is associated with a complex cell cycle dysregulation (CCD) that involves increased activation of the cyclin B/p34-cdc2 complex and abnormal nucleolar structure with dysregulation of nucleolin turnover. Here we report that CCD is also present during pathogenic SIV infection of RMs, and its extent correlates with the level of immune activation and T-cell apoptosis. In marked contrast, naturally SIV-infected SMs show normal regulation of cell cycle control (i.e., normal intracellular levels of cyclin B and preserved nucleolin turnover) and a low propensity to apoptosis in both peripheral blood- and lymph node-derived T cells. The absence of significant CCD in the AIDS-free, non-immune-activated SMs despite high levels of viral replication indicates that CCD is a marker of disease progression during lentiviral infection and supports the hypothesis that the preservation of cell cycle control may help to confer the disease-resistant phenotype of SIV-infected SMs.

Transient upregulation of connexin43 gap junctions and synchronized cell cycle control precede myoblast fusion in regenerating skeletal muscle in vivo

The spatio-temporal expression of gap junction connexins (Cx) was investigated and correlated with the progression of cell cycle control in regenerating soleus muscle of Wistar rats. Notexin caused a selective myonecrosis followed by the complete recapitulation of muscle differentiation in vivo, including the activation, commitment, proliferation, differentiation and fusion of myogenic cells. In regenerating skeletal muscle, only Cx43 protein, out of Cx-s 26, -32, -37, -40, -43 and -45, was detected in desmin positive cells. Early expression of Cx43 in the proliferating single myogenic progenitors was followed by a progressive upregulation in interacting myoblasts until syncytial fusion, and then by a rapid decline in multinucleate myotubes. The significant upregulation of Cx43 gap junctions in aligned myoblasts preceding fusion was accompanied by the widespread nuclear expression of cyclin-dependent kinase inhibitors p21(waf1/Cip1) and p27(kip1) and the complete loss of Ki67 protein. The synchronized exit of myoblasts from the cell cycle following extensive gap junction formation suggests a role for Cx43 channels in the regulation of cell cycle control. The potential of Cx43 channels to stimulate p21(waf1/Cip1) and p27(kip1) is known. In the muscle, proving the involvement of Cx43 in either a direct or a bystander cell cycle regulation requires functional investigations.

Role of the retinoblastoma tumor suppressor protein in cellular differentiation

The retinoblastoma protein (pRb105) is a true tumor suppressor as deregulation of the Rb pathway by either mutation of pRb105 itself or other proteins in the pathway, such as p16INK4a, occur in most cancers. This prototypical family member, along with the related p107 and p130, are involved in the control of cell cycle regulation, but pRb105 has also been shown to be involved in tissue development and differentiation. This prospective will discuss the increasing evidence for a role of pRb105 in cellular differentiation and the fact that various cancers, which contain mutant pRb105, or mutations in proteins in the pRb105 pathway, are perhaps a result of deregulation of differentiation.

The Enigmatic X Gene of Hepatitis B Virus

The exact function of HBx during HBV replication and its influence in HBV-associated hepatocellular carcinoma are still undefined, but common themes are emerging. Most studies agree that, if not absolutely essential for HBV infections, HBx at the very least plays a critical role in viral replication. HBx functions in the cytoplasm to activate various signaling pathways, many of which are controlled by modulation of cytosolic calcium. A number of groups have now demonstrated that HBx activities somehow involve modulation of cytosolic calcium, although the exact molecular mechanism is undefined. In the nucleus, HBx can regulate transcription through a direct interaction with different transcription factors and, in some cases, enhance their binding to specific transcription elements. Clearly, HBx can influence apoptotic and cell cycle regulatory pathways, but the exact consequences of these activities of HBx are likely affected by the cell types in which the studies have been conducted. In this regard, it is interesting to note that much of the seeming confusion or controversy regarding HBx activities is likely the result of the different assays performed, and the findings of different studies may ultimately be more compatible than expected. In most experiments, HBx activities have been analyzed based on the end product of what are long and complex pathways. For example, transcriptional reporter readout, apoptotic assays, cell cycle regulation, and mutagenic studies are judged based on an analysis of the final product of a given pathway without considering common effectors. HBx may initiate overlapping and very similar effectors in all the studies conducted, but the consequence of this will likely vary depending on the specific cellular phenotype and the assay involved. Moreover, the frequency or longevity of this stimulus may vary depending on the amount of HBx present, which in turn influences the results. Fluctuations in the frequency or longevity of a HBx-induced stimulus, such as calcium signaling, can have a dramatic influence on the final effect on a cell (reviewed in reference 19). Equally interesting is the possibility that HBx has different consequences for hepatocyte physiology as HBV-infected cells are targeted by the immune system or as hepatocytes in which HBx is expressed undergo transformation and progression to HCC. Both these processes are dynamic, and it is likely that cellular physiology is consequently altered. The demonstration that different signaling pathways can be activated based on the differentiation state of hepatocytes derived from the same parental cell line and suggestions that the cellular status of p53, p21, and p27 can affect HBx-dependent regulation of cell cycle control supports the notion that the consequence of HBx expression may change as hepatocytes become transformed (2, 62, 93). It remains to be determined whether similar results will be observed in the context of an authentic HBV infection. Such observations will add to the complexity of identifying and understanding the precise molecular mechanism of HBx activities during viral replication. However, there is the exciting possibility that the end product of HBx activities may change as the cell responds to the infection. Finally, studies of HBx activities during HBV replication should help clarify the many properties that have been ascribed to HBx expression, including those that are required for viral replication and those that may influence cellular transformation.

PE-1/METS, an Antiproliferative Ets Repressor Factor, Is Induced by CREB-1/CREM-1 during Macrophage Differentiation

The molecular mechanisms involved in regulating the balance between cellular proliferation and differentiation remain poorly understood. Members of the Ets-domain family of transcription factors are candidates for proteins that might differentially regulate cell cycle control and cell type-specific genes during the differentiation of myeloid progenitor cells. The Ets repressor PE-1/METS has been suggested to contribute to growth arrest during terminal macrophage differentiation by repressing Ets target genes involved in Ras-dependent proliferation. An important feature of this regulatory model is that PE-1/METS is itself induced by the program of macrophage differentiation elicited by M-CSF. Here, we present evidence that the PE-1/METS gene is a transcriptional target of the cyclic AMP response element-binding protein-1 (CREB-1). CREB-1 expression is dramatically up-regulated during macrophage differentiation and phosphorylation of CREB-1 and the related factor CREM-1 are stimulated by M-CSF in a SAPK2/p38-dependent manner. Chromatin immunoprecipitation experiments demonstrate that CREB-1/CREM-1 are recruited to the PE-1/METS promoter as well as to the promoters of other genes that are up-regulated during terminal macrophage differentiation. Overexpression of CREB-1 stimulates the activities of the PE-1/METS, and macrosialin promoters, while expression of a dominant negative form of CREB-1 during macrophage differentiation inhibits expression of the PE-1/METS and macrosialin genes. Inhibition of CREB function also results in reduced expression of CD54 and impaired cell adhesion. Taken together, these findings reveal new roles of CREB-1/CREM-1 as regulators of macrophage differentiation.

Radiation response of neural precursor cells: linking cellular sensitivity to cell cycle checkpoints, apoptosis and oxidative stress.

Therapeutic irradiation of the brain can cause a progressive cognitive dysfunction that may involve defects in neurogenesis. In an effort to understand the mechanisms underlying radiation-induced stem cell dysfunction, neural precursor cells isolated from the adult rat hippocampus were analyzed for acute (0-24 h) and chronic (3-33 days) changes in apoptosis and reactive oxygen species (ROS) after exposure to X rays. Irradiated neural precursor cells exhibited an acute dose-dependent apoptosis accompanied by an increase in ROS that persisted over a 3-4-week period. The radiation effects included the activation of cell cycle checkpoints that were associated with increased Trp53 phosphorylation and Trp53 and p21 (Cdkn1a) protein levels. In vivo, neural precursor cells within the hippocampal dentate subgranular zone exhibited significant sensitivity to radiation. Proliferating precursor cells and their progeny (i.e. immature neurons) exhibited dose-dependent reductions in cell number. These reductions were less severe in Trp53-null mice, possibly due to the disruption of apoptosis. These data suggest that the apoptotic and ROS responses may be tied to Trp53-dependent regulation of cell cycle control and stress-activated pathways. The temporal coincidence between in vitro and in vivo measurements of apoptosis suggests that oxidative stress may provide a mechanistic explanation for radiation-induced inhibition of neurogenesis in the development of cognitive impairment.

Defective endochondral ossification in mice with strongly compromised expression of JunB.

Functional analysis in mice has established an absolute requirement of JunB, a member of the AP-1 transcription factor family, during early embryonic development. To investigate the role of JunB during mid and late gestation and postnatally Ubi-junB transgenic mice were used to generate two junB-/- Ubi-junB mutant lines, in which embryonic lethality was rescued but strongly reduced JunB expression in several adult tissues was observed. Mutant mice from both rescue lines were growth retarded and shared significantly reduced longitudinal bone growth. Mutant long bones were characterised by reduced numbers of growth plate chondrocytes and a severe osteoporosis. Decreased JunB levels in epiphysal growth plate chondrocytes and bone lining osteoblasts correlated with deregulated expression of Cyclin A, Cyclin D1 and p16INK4a, key regulators of cell cycle control. Furthermore, junB-/- Ubi-junB bone marrow stromal cells were unable to differentiate into bone forming osteoblasts in vitro. Our data demonstrate that JunB plays a crucial role in endochondral ossification by regulating proliferation and function of chondrocytes and osteoblasts.

IFI16 as a Negative Regulator in the Regulation of p53 and p21Waf1

IFI16 is a member of the HIN-200 family (hematopoietic interferon-inducible nuclear antigens with 200 amino acid repeats) that contains a DNA binding domain, a transcriptional regulatory domain, and DAPIN/PAAD, a protein domain associated with interferon response. It can function as a transcription repressor and directly binds p53. Although the structural and biochemical properties of IFI16 are known, the physiological relevance of these properties in the cellular context is still elusive. Here we report that the inhibition of endogenous IFI16 expression by small interfering RNA (siRNA) induces p21Waf1 mRNA and protein expression through p53 but does not induce pro-apoptotic p53 target genes. This rapid induction of p21 was wild-type p53-dependent and resulted in cell cycle arrest along with a marked reduction of phosphorylated Rb in normally growing cells. We also showed that the repression of IFI16 affects p53 transcriptional activity at the p21 promoter as well as the protein stability of p53 and p21. Our findings identified a new role for IFI16 in modulating p53 function and its target gene regulation in the control of cell cycle regulation.

CELL CYCLE GENE EXPRESSION DURING SKELETAL MUSCLE GROWTH

PURPOSE Skeletal muscle growth, under some conditions, is thought to be dependent on the proliferation of satellite cells. The proliferation of satellite cells is dependent on the interaction of a number of proteins involved with the control of the cell cycle. The objective of this study was to test the hypothesis that the longitudinal growth of skeletal muscle results in the upregulation of mRNA levels of key genes responsible for promoting the cell cycle. METHODS Female Sprague-Dawley rats were assigned to the following groups: 1) control (CON; n =14); 2) frame-control; (FC; n=6); and 3) tibial osteotomy and distraction; (DIST; n = 8). Following 8 days of tibial distraction (lengthening), the soleus muscles were harvested and used for oligonucleotide microarray analyses (Affymetrix; RGU34A chipset). Analyses of the microarray data were performed using a statistical package (dCHIP) in combination with an analytical/visualization software package (GeneSpring). The results from these analyses were then integrated into a pathway for cell cycle control obtained from the Kyoto Encyclopedia of Genes and Genomes (www.kegg.com). RESULTS Approximately 30 different genes associated with the cell cycle were examined. Interestingly, the mRNA levels that were upregulated the most belonged to genes (GADD45, p21, PCNA, rb) that promote cell cycle arrest. Supplemental analyses of GADD45 (growth arrest and DNA destruction) isoforms (alpha, beta, and gamma) via RTPCR revealed significant increases (P < 0.05) in the alpha and beta isoforms. CONCLUSION The findings of this study are paradoxical to the satellite cell hypothesis, which states that skeletal muscle growth is associated with proliferation of satellite cells. It may be that the changes observed in the mRNA levels in the current study are not reflective of the protein-protein interactions involved in the regulation of cell cycle control. Funded by NIH AR46856 (VJC), AR44743 (VJC), AR45594 (GRA), and AR30346 (KMB)

Sonic hedgehog-dependent activation of Gli2 is essential for embryonic hair follicle development.

Sonic hedgehog (Shh) signaling plays a critical role in hair follicle development and skin cancer, but how it controls these processes remains unclear. Of the three Gli transcription factors involved in transducing Shh signals in vertebrates, we demonstrate here that Gli2 is the key mediator of Shh responses in skin. Similar to Shh(-/-) mice, Gli2(-/-) mutants exhibit an arrest in hair follicle development with reduced cell proliferation and Shh-responsive gene expression, but grossly normal epidermal differentiation. By transgenic rescue experiments, we show that epidermal Gli2 function alone is sufficient to restore hair follicle development in Gli2(-/-) skin. Furthermore, only a constitutively active form of Gli2, but not wild-type Gli2, can activate Shh-responsive gene expression and promote cell proliferation in Shh(-/-) skin. These observations indicate that Shh-dependent Gli2 activator function in the epidermis is essential for hair follicle development. Our data also reveal that Gli2 mediates the mitogenic effects of Shh by transcriptional activation of cyclin D1 and cyclin D2 in the developing hair follicles. Together, our results suggest that Shh-dependent Gli2 activation plays a critical role in epithelial homeostasis by promoting proliferation through the transcriptional control of cell cycle regulators.

The pharmaceutical industry and research in 2002 and beyond.

The success of the pharmaceutical industry will continue to depend on its ability to satisfy the clinical needs of established market economies. The number and quality of new drugs emerging from development pipelines seems likely to rise due to increased research and development budgets of the merged pharmaceutical companies, efficiencies across all facets of the development process, increasing use of new technologies and availability of new targets from the ongoing work on the role of human genes in disease pathways. In addition to the traditional small-molecule drugs, the market for protein products, including monoclonal antibodies and therapeutic vaccines, is likely to expand as advances in recombinant and formulation technologies are made. Current work on relatively newer fields of pharmaceutical research, such as novel G-protein-coupled receptors, chemokines/cytokines, integrins and control of cell cycle regulation and signal transduction pathways (kinases, phosphatases and transcription factors) will lead to new drugs over the next decade. It is tempting to argue that a progressive fall in the number of new drugs in the last decade of the 20th century reflects the end of an era as companies struggle to identify any remaining quality products using old-style drug hunting practices.

Assays for cyclin-dependent kinase inhibitors.

Cyclin-dependent kinases (CDK) are serine/threonine kinases that regulate cell cycle control and progression (,). A CDK holoenzyme complex is active if associated with its cyclin partner and if the complex is phosphorylated at specific activating residues (threonine 160/161) (,). The progression through the cell cycle is mediated by the sequential activation of CDKs during different phases of the cycle. The G1/S phase transition of the cell cycle is mediated by phosphorylation of the Retinoblastoma gene product (Rb) by CDK4 and/or CDK6 (G1/S checkpoint), leading to the release of bound E2F from Rb and allowing the transcription of genes by “free E2F” necessary for S phase progression. During late G1 phase, CDK2/cyclin E complex phosphorylates several substrates including Rb. On the other hand, CDK2/cyclin A complex is active throughout S phase. For cells to progress to mitosis, CDK1 (CDC2) must be “activated,” and activated cdc2 phosphorylates substrates required for mitosis ().

A two-stage, p16(INK4A)- and p53-dependent keratinocyte senescence mechanism that limits replicative potential independent of telomere status.

With increasing frequency during serial passage in culture, primary human keratinocytes express p16(INK4A) (p16) and undergo senescence arrest. Keratinocytes engineered to express hTERT maintain long telomeres but typically are not immortalized unless, by mutation or other heritable event, they avoid or greatly reduce p16 expression. We have confirmed that keratinocytes undergo p16-related senescence during growth in culture, whether in the fibroblast feeder cell system or in the specialized K-sfm medium formulation, and that this mechanism can act as a barrier to immortalization following hTERT expression. We have characterized the p16-related arrest mechanism more precisely by interfering specifically with several regulators of cell cycle control. Epidermal, oral mucosal, corneal limbal, and conjunctival keratinocytes were transduced to express a p16-insensitive mutant cdk4 (cdk4(R24C)), to abolish p16 control, and/or a dominant negative mutant p53 (p53DD), to abolish p53 function. Expression of either cdk4(R24C) or p53DD alone had little effect on life span, but expression of both permitted cells to divide 25 to 43 population doublings (PD) beyond their normal limit. Keratinocytes from a p16(+/-) individual transduced to express p53DD alone displayed a 31-PD life span extension associated with selective growth of variants that had lost the wild-type p16 allele. Cells in which both p53 and p16 were nonfunctional divided rapidly during their extended life span but experienced telomere erosion and ultimately ceased growth with very short telomeres. Expression of hTERT in these cells immortalized them. Keratinocytes engineered to express cdk4(R24C) and hTERT but not p53DD did not exhibit an extended life span. Rare immortal variants exhibiting p53 pathway defects arose from them, however, indicating that the p53-dependent component of keratinocyte senescence is telomere independent. Mutational loss of p16 and p53 has been found to be a frequent early event in the development of squamous cell carcinoma. Our results suggest that such mutations endow keratinocytes with extended replicative potential which may serve to increase the probability of neoplastic progression.

Nuclear to cytoplasmic compartment shift of the p33ING1b tumour suppressor protein is associated with malignancy in melanocytic lesions.

Cutaneous malignant melanoma is an unpredictable neoplasm. Studies of cell cycle and proliferation-associated proteins may help in the understanding of the genesis of melanomas. The tumour suppressor gene TP53 has been shown to be involved in melanomas. However, the incidence of TP53 malfunction in cutaneous melanoma is unclear, and other regulators of cell cycle control are likely to be involved in both the development and progression of melanocytic neoplasia. A candidate is the ING1 gene, which co-operates with TP53 in growth suppression and apoptosis. Thus loss of ING1 function may have similar consequences to loss of TP53 function and may contribute to tumorigenesis. Therefore we have studied the expression of p33ING1b protein in cutaneous melanocytic neoplasia. Sixty-seven melanocytic lesions were studied by immunohistochemistry for the expression of p33ING1b. In our series there was loss of nuclear p33ING1b expression in invasive malignant melanoma compared with normal cutaneous melanocytes or the melanocytes of benign melanocytic naevi. This was associated with an enhancement of cytoplasmic p33ING1b expression which was particularly prominent in invasive malignant melanoma. Cytoplasmic immunostaining for p33ING1b using MAb GN2 is strongly associated with 'activated' melanocytic lesions; therefore it is possible that this MAb could be of value in diagnostic practice. Furthermore, the reduction in p33ING1b expression and perhaps translocation from the nucleus to the cytoplasm may play a central role in the development and progression of melanomas.

Age-related mutation accumulation at a lacZ reporter locus in normal and tumor tissues of Trp53-deficient mice

Increased genomic instability has been found associated with cancer and aging. The p53 tumor suppressor protein is a major determinant of genomic instability as a regulator of cell cycle control and apoptosis in response to DNA damage. To investigate the rate of age-related mutation accumulation in the absence of p53, we crossed Trp53 null mice with transgenic mice harboring a lacZ mutational target gene. In the hybrid animals, lacZ mutation frequencies at early age (i.e. at about 2 months) were found to be the same as in the control lacZ animals. However, up until about 6 months, when the Trp53-knockout mice usually die from cancer, mutations were found to accumulate with age in the spleen, and to a lesser extent in the liver, at a more rapid rate than in the control Trp53 +/+ or Trp53 +/−, lacZ hybrid mice. Treatment of 2–3-month-old Trp53 −/−, lacZ hybrid mice with the powerful mutagen ethyl nitrosourea (ENU) resulted in a higher number of mutations induced in the liver but not in the spleen, as compared to the Trp53 +/+, lacZ mice. These results suggest that p53 is not an important determinant of gene mutation induction, either spontaneously during development or after treatment with a mutagen. The accelerated age-related accumulation of mutations in normal spleen and liver could be explained by the defect in apoptosis, which would prevent severely damaged cells from being eliminated.