Downregulation Of Cyclin-dependent Kinase Inhibitor Research Articles

Loss of fragile WWOX gene leads to senescence escape and genome instability

Induction of DNA damage response (DDR) to ensure accurate duplication of genetic information is crucial for maintaining genome integrity during DNA replication. Cellular senescence is a DDR mechanism that prevents the proliferation of cells with damaged DNA to avoid mitotic anomalies and inheritance of the damage over cell generations. Human WWOX gene resides within a common fragile site FRA16D that is preferentially prone to form breaks on metaphase chromosome upon replication stress. We report here that primary Wwox knockout (Wwox−/−) mouse embryonic fibroblasts (MEFs) and WWOX-knockdown human dermal fibroblasts failed to undergo replication-induced cellular senescence after multiple passages in vitro. Strikingly, by greater than 20 passages, accelerated cell cycle progression and increased apoptosis occurred in these late-passage Wwox−/− MEFs. These cells exhibited γH2AX upregulation and microsatellite instability, indicating massive accumulation of nuclear DNA lesions. Ultraviolet radiation-induced premature senescence was also blocked by WWOX knockdown in human HEK293T cells. Mechanistically, overproduction of cytosolic reactive oxygen species caused p16Ink4a promoter hypermethylation, aberrant p53/p21Cip1/Waf1 signaling axis and accelerated p27Kip1 protein degradation, thereby leading to the failure of senescence induction in Wwox-deficient cells after serial passage in culture. We determined that significantly reduced protein stability or loss-of-function A135P/V213G mutations in the DNA-binding domain of p53 caused defective induction of p21Cip1/Waf1 in late-passage Wwox−/− MEFs. Treatment of N-acetyl-l-cysteine prevented downregulation of cyclin-dependent kinase inhibitors and induced senescence in Wwox−/− MEFs. Our findings support an important role for fragile WWOX gene in inducing cellular senescence for maintaining genome integrity during DDR through alleviating oxidative stress.

Single or combined ablation of peripheral serotonin and p21 limit adipose tissue expansion and metabolic alterations in early adulthood in mice fed a normocaloric diet.

Identifying the fundamental molecular factors that drive weight gain even in the absence of hypercaloric food intake, is crucial to enable development of novel treatments for the global pandemic of obesity. Here we investigated both adipose tissue-specific and systemic events that underlie the physiological weight gain occurring during early adulthood in mice fed a normocaloric diet. In addition, we used three different genetic models to identify molecular factors that promote physiological weight gain during normocaloric and hypercaloric diets. We demonstrated that normal physiological weight gain was accompanied by an increase in adipose tissue mass and the presence of cellular and metabolic signatures typically found during obesity, including adipocyte hypertrophy, macrophage recruitment into visceral fat and perturbed glucose metabolism. At the molecular level, this was associated with an increase in adipose tissue tryptophan hydroxylase 1 (Tph1) transcripts, the key enzyme responsible for the synthesis of peripheral serotonin. Genetic inactivation of Tph1 was sufficient to limit adipose tissue expansion and associated metabolic alterations. Mechanistically, we discovered that Tph1 inactivation resulted in down-regulation of cyclin-dependent kinase inhibitor p21Waf1/Cip1 expression. Single or double ablation of Tph1 and p21 were equally effective in preventing adipocyte expansion and systemic perturbation of glucose metabolism, upon both normocaloric and hypercaloric diets. Our results suggest that serotonin and p21 act as a central molecular determinant of weight gain and associated metabolic alterations, and highlights the potential of targeting these molecules as a pharmacologic approach to prevent the development of obesity.

The Metabolite R-2-Hydroxyglutarate (R2HG) Collaborates with HoxA9 to Induce Monocytic Leukemia

Mutations in the metabolic enzymes IDH1 and IDH2 are frequently found in several tumors including glioma and acute myeloid leukemia (AML). Mutant IDH produces R-2-hydroxyglutarate (R2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators, thus linking metabolism to tumorigenesis. However, it is unknown whether R2HG alone is sufficient to recapitulate the biologic effects of mutant IDH1 in vivo. Recently, we have shown that IDH1mut cooperates with HoxA9 and induces a monocytic leukemia in mice. In order to evaluate the effects of R2HG independently of the mutated IDH1 protein and to determine whether the effects are specific to the R-enantiomer of 2HG, we treated mice transplanted with HoxA9 immortalised bone marrow cells with R2HG, S-2-hydroxyglutarate (S2HG), alpha-ketoglutarate (aKG) and phosphate buffered saline (PBS). The mice in the metabolite cohorts received an intraperitoneal dose of 1 mg per day.Mice treated with R2HG had higher engraftment levels at 16 and 20 weeks post transplantation than the mice treated with S2HG, αKG and PBS respectively (P<.01). High WBC counts (70±16 /nl) and lower platelet counts than in control mice were observed in the cohort receiving R2HG after 16 to 20 weeks of treatment, while the S2HG, αKG and PBS cohorts had normal blood counts even at 20 weeks (P<.05). Peripheral blood from R2HG treated mice revealed significantly more immature Mac1+Gr1- and less mature Mac1+Gr1+ cells at 12 and 16 weeks after treatment than S2HG, αKG and PBS treated mice (P<.001). In addition, the R2HG treated mice died with a median latency of 137 days post transplantation from monocytic leukemia, while mice treated with S2HG, αKG and PBS died with a median latency of 223, 202 and 184 days respectively (P<.001). Further, in order to assess whether R2HG alone was sufficient as a single hit to induce myeloproliferation, normal C57BL/6 mice (without HoxA9) were treated with R2HG, S2HG and PBS for eight months. No differences were observed for survival, blood counts, immunophenotype and frequencies of progenitor cells (lin-ckit+sca1+, CMP, GMP and MEP) between treatment groups and control. This data shows that the metabolite R2HG like the IDH1 mutant protein cooperates with HoxA9 to induce monocytic leukemia.We next compared mice receiving transplants of HoxA9+IDH1mut cells with mice receiving HoxA9 cells that were then treated with R2HG. Both cohorts developed monocytic leukemia, albeit with different kinetics. The Hoxa9+IDH1mut mice died with a median latency of 83 days while the R2HG cohort died with a median latency of 137 days post transplantation (P<0.001). Also, while the former cohort developed severe leukocytosis, anemia and thrombocytopenia at 12 weeks, the R2HG treated mice had high WBC counts and lower platelet counts than control mice at 16 to 20 weeks after treatment. The faster disease kinetics in IDH1mut mice could be attributed to a significantly lower proportion of cells in G0/G1 and higher proportion of cells in S phase when compared to cells from mice treated with R2HG at 9 weeks after transplantation (P<.001). This resulted from a marked downregulation of cyclin-dependent kinase inhibitors (Cdkn) 1A (p21), 1B (p27), 2A (p16), and 2B (p15) in HoxA9+IDH1mut cells as compared to HoxA9 cells treated with R2HG or PBS.In order to rule out an influence of differential R2HG levels on the disease progression between the two cohorts, levels of R2HG were quantified. IDH1mut expressing and R2HG treated bone marrow cells from mice had similarly high ratios of R2HG/S2HG. The quantified R2HG levels were also comparable to that of primary AML patient cells harbouring mutated IDH1. In unsupervised hierarchical clustering mutated cells clustered together with R2HG and separated from PBS treated mice. 69 of the top 100 enriched Gene Ontology gene sets of HoxA9+IDH1mut were also found in HoxA9+R2HG, suggesting largely redundant but also non-overlapping functions of the mutant IDH1 protein and the oncometabolite R2HG.In summary, we show that R2HG, similar to the mutant IDH1 protein, promotes leukemogenesis in cooperation with HoxA9, although with delayed kinetics. Our data proves that R2HG acts as an oncometabolite in vivo in a murine model of leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

FHL2 regulates hematopoietic stem cell functions under stress conditions.

FHL2, a member of the four and one half LIM domain protein family, is a critical transcriptional modulator. Here, we identify FHL2 as a critical regulator of hematopoietic stem cells (HSCs) that is essential for maintaining HSC self-renewal under regenerative stress. We find that Fhl2 loss has limited effects on hematopoiesis under homeostatic conditions. In contrast, Fhl2-null chimeric mice reconstituted with Fhl2-null bone marrow cells developed abnormal hematopoiesis with significantly reduced numbers of HSCs, hematopoietic progenitor cells (HPCs), red blood cells and platelets as well as hemoglobin levels. In addition, HSCs displayed a significantly reduced self-renewal capacity and were skewed toward myeloid lineage differentiation. We find that Fhl2 loss reduces both HSC quiescence and survival in response to regenerative stress, probably as a consequence of Fhl2-loss-mediated down-regulation of cyclin dependent kinase (CDK)-inhibitors, including p21(Cip) and p27(Kip1). Interestingly, FHL2 is regulated under control of a tissue specific promoter in hematopoietic cells and it is down-regulated by DNA hypermethylation in the leukemia cell line and primary leukemia cells. Furthermore, we find that down-regulation of FHL2 frequently occurs in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients, raising a possibility that FHL2 down-regulation plays a role in the pathogenesis of myeloid malignancies.

Berberine attenuates high glucose-induced proliferation and extracellular matrix accumulation in mesangial cells: Involvement of suppression of cell cycle progression and NF-κB/AP-1 pathways

Berberine has been shown to have renoprotective effects on diabetes through attenuating TGF-β1 and fibronectin (FN) expression. However, how berberine regulates TGF-β1 and FN is not fully clear. Here we investigated whether berberine inhibited TGF-β1 and FN expression in high glucose-cultured mesangial cells. Berberine significantly inhibited mesangial cell proliferation and hypertrophy by increasing the cell population in G1-phase and reducing that in S-phase. In addition, berberine reversed high glucose-induced down-regulation of cyclin-dependent kinase inhibitor p21Waf1/Cip1 and p27Kip1. Berberine inhibited p65 translocation to the nucleus and c-jun phosphorylation induced by high glucose. Furthermore, berberine attenuated high glucose-induced expression of TGF-β1 and FN. Using a luciferase reporter assay, we found that high glucose-induced transcription activity of NF-κB and AP-1 was blocked by berberine. Electrophoretic mobility shift assay showed that high glucose increased that NF-κB and AP-1 DNA binding activity. These data indicate that berberine inhibited mesangial cell proliferation and hypertrophy by modulating cell cycle progress. In addition, berberine suppressed high glucose-induced TGF-β1 and FN expression by blocking NF-κB/AP-1 pathways.

Mortalin and DJ-1 coordinately regulate hematopoietic stem cell function through the control of oxidative stress

AbstractHematopoietic stem cells (HSCs) maintain stemness through various mechanisms that protect against stressful conditions. Heat shock proteins (HSPs) preserve cell homeostasis during stress responses through protein quality control, suggesting that HSPs may safeguard HSCs against numerous traumas. Here, we show that mortalin, a mitochondrial HSP, plays an essential role in maintaining HSC properties by regulating oxidative stress. Mortalin is primarily localized in hematopoietic stem and progenitor cell (HSPC) compartments. In this study, the inhibition of mortalin function caused abnormal reactive oxygen species (ROS) elevation in HSCs and reduced HSC numbers. Knockdown (KD) of mortalin in HSPCs impaired their ability to repopulate and form colonies. Moreover, mortalin-KD HSCs could not maintain quiescence and showed severe downregulation of cyclin-dependent kinase inhibitor– and antioxidant-related genes. Conversely, HSCs that overexpressed mortalin maintained a high reconstitution capacity and low ROS levels. Furthermore, DJ-1, one of the genes responsible for Parkinson's disease, directly bound to mortalin and acted as a negative ROS regulator. Using DJ-1–deficient mice, we demonstrated that mortalin and DJ-1 coordinately maintain normal ROS levels and HSC numbers. Collectively, these results indicate that the mortalin/DJ-1 complex guards against mitochondrial oxidative stress and is indispensable for the maintenance of HSCs.

Upregulation of miR-370 contributes to the progression of gastric carcinoma via suppression of FOXO1

FOXO1 is downregulated in a number of cancers. However, the underlying mechanisms are poorly understood. In this study, we report that the expression of miR-370 was upregulated in gastric cancer cell lines and gastric cancer tissues. Overexpression of miR-370 in gastric cancer cells promoted the cell proliferation and anchorage-independent growth, while silencing of miR-370 showed opposite effects. miR-370-induced proliferation was correlated with the downregulation of cyclin-dependent kinase inhibitors, p27Kip1 and p21Cip1, and the upregulation of the cell cycle regulator cyclin D1. Furthermore, we identified that FOXO1 is the functional target of miR-370. Restored expression of FOXO1 together with miR-370 strongly abrogated miR-370-induced cell proliferation. Taken together, our results revealed a novel mechanism of FOXO1 suppression mediated by miR-370 in gastric cancer.

Doxycycline Regulated Induction of AKT in Murine Prostate Drives Proliferation Independently of p27 Cyclin Dependent Kinase Inhibitor Downregulation

The PI3 kinase/AKT pathway has been shown to increase degradation of the p27 cyclin dependent kinase inhibitor through phosphorylation of consensus AKT sites on p27 and SKP2, and AKT driven proliferation may be checked by feedback mechanisms that increase p27 expression and induce senescence. However, these AKT sites are not conserved in mouse, and it has not been clear whether AKT negatively regulates murine p27. Transgenic mice with a probasin promoter controlled prostate specific reverse tetracycline transactivator (ARR2Pb-rtTA) were generated and used to achieve doxycycline inducible expression of a tetracycline operon regulated constitutively active myristoylated AKT1 transgene (tetO-myrAKT). Doxycycline induction of myrAKT occurred within 1 day and rapidly induced proliferation (within 4 days) and the development of prostatic intraepithelial neoplasia (PIN) lesions in ventral prostate, which did not progress to prostate cancer. Cells in these lesions expressed high levels of p27, had increased proliferation, and there was apoptosis of centrally located cells. Doxycycline withdrawal resulted in apoptosis of cells throughout the lesions and rapid clearing of hyperplastic glands, confirming in vivo the critical antiapoptotic functions of AKT. Significantly, analyses of prostates immediately after initiating doxycycline treatment further showed that p27 expression was rapidly increased, coincident with the induction of myrAKT and prior to the development of hyperplasia and PIN. These findings establish in vivo that murine p27 is not negatively regulated by AKT and indicate that proliferation in PI3 kinase/AKT pathway driven mouse models is mediated by p27 independent mechanisms that may be distinct from those in human. Further studies using prostate specific doxycycline regulated transgene expression may be useful to assess the acute effects of inducing additional transgenes in adult murine prostate epithelium, and to assess the requirements for continued transgene expression in transgene induced tumors.

Retinoblastoma tumor-suppressor protein phosphorylation and inactivation depend on direct interaction with Pin1

Inactivation of the retinoblastoma protein (pRb) by phosphorylation triggers uncontrolled cell proliferation. Accordingly, activation of cyclin-dependent kinase (CDK)/cyclin complexes or downregulation of CDK inhibitors appears as a common event in human cancer. Here we show that Pin1 (protein interacting with NIMA (never in mitosis A)-1), a peptidylprolyl isomerase involved in the control of protein phosphorylation, is an essential mediator for inactivation of the pRb. Our results indicate that Pin1 controls cell proliferation by altering pRb phosphorylation without affecting CDK and protein phosphatase 1 and 2 activity. We demonstrated that Pin1 regulates tumor cell proliferation through direct interaction with the spacer domain of the pRb protein, and allows the interaction between CDK/cyclin complexes and pRb in mid/late G1. Phosphorylation of pRb Ser 608/612 is the crucial motif for Pin1 binding. We propose that Pin1 selectively boosts the switch from hypo- to hyper-phosphorylation of pRb in tumor cells. In addition, we demonstrate that the CDK pathway is responsible for the interaction of Pin1 and pRb. Prospectively, our findings therefore suggest that the synergism among CDK and Pin1 inhibitors holds great promise for targeted pharmacological treatment of cancer patients, with the possibility of reaching high effectiveness at tolerated doses.

Significance of p57Kip2 Down-Regulation in Oncogenesis of Bladder Carcinoma: An Immunohistochemical Study

Cyclin-dependent kinase inhibitors have important roles in the oncogenesis of various tumors including urothelial cancer. The aim of this study was to establish the importance of p57(Kip2), a unique cyclin-dependent kinase inhibitor, in the oncogenesis of bladder carcinoma. This article also focused on another cyclin-dependent kinase inhibitor, p27(Kip1), and telomerase enzyme and examined the relationship between these proteins. Thirty-one patients with urothelial carcinomas of the bladder and 7 cases with normal urinary bladder mucosa were included in the study. Immunohistochemical study was performed by monoclonal antibodies of p27(Kip1), p57(Kip2), and the telomerase subunit (hTERT). All immunohistochemical preparations were evaluated by an immunohistochemical histological score. p57(Kip2) and p27Kip1) expression were seen in all of the cases of normal mucosa. In carcinoma cases, 8 of 31 (25.8%) showed p57(Kip2) nuclear positivity and 20 of 31 (64.5%) expressed nuclear p27(Kip1). HSCOREs of carcinoma cases showed lower scores of nuclear p57(Kip2) and p27(Kip1) than normal mucosa, but only HSCOREs of nuclear p57(Kip2) (P = 0.001) showed statistical significance. Despite unknown significance, cytoplasmic p57(Kip2) and p27(Kip1) were also evaluated. Immunohistochemical analysis showed that carcinomas expressed higher HSCOREs of hTERT than normal mucosa, and there was a significant difference (P = 0.026) between muscle invasive carcinomas and normal mucosa. The data showed that p57(Kip2) down-regulation along with p27(Kip1) is a well-established feature of urothelial carcinoma. Probably, this down-regulation of cyclin-dependent kinase inhibitors supports the proliferation phase of oncogenesis. In the study, we also showed that hTERT expression was up-regulated in higher stages of urothelial carcinoma.

Genistein Inhibits Rat Aortic Smooth Muscle Cell Proliferation Through the Induction of p27kip1

Diet is one of the most important factors that influence the risks for cardiovascular diseases. Genistein, an isoflavone found in soy, may benefit the cardiovascular system. Here, we investigated the effect of genistein on platelet-derived growth factor (PDGF)-BB–induced proliferation of primary cultured rat aortic smooth muscle cells (RASMCs). Genistein significantly inhibited 25 ng/ml PDGF-BB–induced RASMC proliferation and [3H]-thymidine incorporation into DNA at 10, 20, and 40 μM. In accordance with these findings, genistein blocked the PDGF-BB–inducible progression through G0/G1 to S phase of the cell cycle in synchronized cells. Western blot analysis showed that genistein not only inhibited phosphorylation of retinoblastoma protein (pRb) and expression of cyclin E, cyclin-dependent kinase (CDK) 2, and proliferating cell nuclear antigen (PCNA) protein, but also inhibited downregulation of cyclin-dependent kinase inhibitor (CKI) p27kip1. However, genistein did not affect p21cip1, CDK4, and cyclin D1 expression or early signal transduction through PDGF beta-receptor, extracellular signal-regulated kinases 1/2 (ERK1/2), Akt, and phospholipase C (PLC) γ1 phosphorylation. These results suggest that genistein inhibits PDGF-BB–induced RASMC proliferation via G0/G1 arrest in association with induction of p27kip1, which may contribute to the beneficial effects of genistein on the cardiovascular system.

Hesperetin, a bioflavonoid, inhibits rat aortic vascular smooth muscle cells proliferation by arresting cell cycle

Diet can be one of the most important factors that influence risks for cardiovascular diseases. Hesperetin, a flavonoid present in grapefruits and oranges, is one candidate that may benefit the cardiovascular system. In this study, we have investigated the effect of hesperetin on the platelet-derived growth factor (PDGF)-BB-induced proliferation of primary cultured rat aortic vascular smooth muscle cells (VSMCs). Hesperetin significantly inhibited 50 ng/ml PDGF-BB-induced rat aortic VSMCs proliferation and [(3)H]-thymidine incorporation into DNA at concentrations of 5, 25, 50, and 100 microM. In accordance with these findings, hesperetin revealed blocking of the PDGF-BB-inducible progression through G(0)/G(1) to S phase of the cell cycle in synchronized cells. Western blot showed that hesperetin inhibited not only phosphorylation of retinoblastoma protein (pRb) and expressions of cyclin A, cyclin D, cyclin E, cyclin-dependent kinase 2 (CDK2) as well as proliferating cell nuclear antigen (PCNA) protein, but also downregulation of cyclin-dependent kinase inhibitor (CKI) p27(kip1), while did not affect CKI p21(cip1), p16(INK4), p53, and CDK4 expressions as well as early signaling transductions such as PDGF beta-receptor, extracellular signal-regulated kinase (ERK) 1/2, Akt, p38, and JNK phosphorylation. These results suggest that hesperetin inhibits PDGF-BB-induced rat aortic VSMCs proliferation via G(0)/G(1) arrest in association with modulation of the expression or activation of cell-cycle regulatory proteins, which may contribute to the beneficial effect of grapefruits and oranges on cardiovascular system.

Downregulation of cyclin-dependent kinase inhibitor; p57 kip2, is involved in the cell cycle progression of vascular smooth muscle cells

Immature vascular smooth muscle cells (VSMCs) proliferate responding to extrinsic mitogens and accumulate in neointima after arterial injuries. Cell proliferation is positively regulated by cyclin/cyclin-dependent kinase (CDK) complex and negatively controlled by CDK inhibitors; CKIs such as p27 kip1 and p57 kip2. In this study, embryonic rat thoracic aorta VSMCs; A10 were G0/G1 arrested by serum starvation, re-stimulated with serum, and harvested every four hours. Both CKIs co-expressed in quiescent VSMCs and rapidly diminished by stimulation. Protein level of p27 kip1 was regulated by both transcription and post-transcription, but that of p57 kip2 was mainly by post-transcription. Supplemental overexpression of p57 kip2 inhibited the activations of G1 cyclin/CDKs and subsequent hyperphosphorylations of all three retinoblastoma pocket proteins as well as G1/S transition of cell cycle. Our findings suggest that the downregulations of not only p27 kip1, but also p57 kip2 responding to mitogenic stimulation, play key roles in the cell cycle progression of VSMCs.

Interferon-γ impairs physiologic downregulation of cyclin-dependent kinase inhibitor, p27 Kip1, during G1 phase progression in macrophages

Cell cycle progression of mouse macrophage cells was impaired by interferon-gamma (IFN-γ). The blockage of G1/S transition was associated with diminution of cyclin-dependent kinase-2 (CDK2)-associated kinase activities. The expression of p21 Cip1 was not upregulated by IFN-γ. Instead, the physiologic downregulation of p27 Kip1 necessary for normal cell cycle progression did not take place sufficiently in the presence of IFN-γ. During normal cell cycle progression, the levels of p27 Kip1 were maximal at early G1 and then decreased gradually. In the presence of IFN-γ, however, the levels of p27 Kip1 discontinued to decrease at a late mid G1 point and were consistently as high as, or higher than, levels observed there. The steady, relatively high-level attachment of p27 Kip1 to CDK2 contributed to the insufficient formation of active cyclin/CDK2, possibly deferring cells from entering S phase.

Telomerase activity in relation to p53 status and clinico-pathological parameters in breast cancer.

Cell cycle deregulation can occur at different levels in cancer. In human breast cancer it includes overexpression of cyclins D1 and E, down-regulation of cyclin-dependent kinase inhibitors and inactivation of the retinoblastoma and p53 tumor suppressor proteins. Telomerase activity is strongly associated with an immortal phenotype and expression of telomerase is linked to the cell cycle. We have recently demonstrated a connection between specific cell cycle defects within the pRB pathway and levels of telomerase activity in breast cancer. In the present study, 106 tumors were investigated for p53 gene and protein status. By single strand conformation polymorphism (SSCP) analysis, 15% showed mutations within exons 5-8 and by immunohistochemistry (IHC), 29% were p53 positive. Tumors with a telomerase activity above median (i.e., telomerase(high)) were significantly associated with p53 protein accumulation (p = 0.004), but not with p53 gene mutations. The strongest telomerase expression was found in tumors with p53 protein accumulation. Morphologic grade, estrogen and progesterone receptor expression differed significantly between the telomerase(high) and telomerase(low) groups (p < 0.0001, p = 0.016 and p = 0.046, respectively), but no difference was observed for stage or nodal status. Telomerase(high) tumors were significantly associated with a poor prognosis for node-negative (N0) patients (p = 0.008), but not for node-positive (N+) patients, whereas the opposite was demonstrated for tumors with p53 accumulation. The survival data indicated that telomerase expression has biological importance particularly for N0 tumors, suggesting that telomerase(low) tumors constitute a group of "pre-immortalized" tumors with a good prognosis.

The cell cycle in breast cancer.

Breast cancer is a heterogeneous disease regarding morphology, invasive behavior, metastatic capacity, hormone receptor expression and clinical outcome. For prediction of prognosis, tumor cell kinetics is an important feature, traditionally evaluated by estimation of cell growth-associated parameters such as mitotic index, S-phase fraction and expression of proliferation coupled proteins, for example proliferating cell nuclear antigen (PCNA) and Ki-67 antigen. Recent data indicate that deregulation of the cell cycle can occur at different levels in cancer and that the "deregulation pattern" can be of clinical significance. In the present overview we give a short description of approaches used for cell proliferation assessments, whereafter more recent data on cell cycle deregulation are discussed. Alterations of importance in breast cancer include overexpression of cyclins D1 and E, down-regulation of cyclin-dependent kinase inhibitors, such as p16, and inactivation of the retinoblastoma and p53 tumor suppressor proteins.