Using motor vehicle crash records for injury surveillance and research in agriculture and forestry.

PROBLEM: Fatal injuries in the agriculture, forestry, and fishing sector (AgFF) outweigh those across all sectors in the United States. Transportation-related injuries are among the top contributors to these fatal events. However, traditional occupational injury surveillance systems may not completely capture crashes involving farm vehicles and logging trucks, specifically nonfatal events. METHODS: The study aimed to develop an integrated database of AgFF-related motor-vehicle crashes for the southwest (Arkansas, Louisiana, New Mexico, Oklahoma, and Texas) and to use these data to conduct surveillance and research. Lessons learned during the pursuit of these aims were cataloged. Activities centered around the conduct of traditional statistical and geospatial analyses of structured data fields and natural language processing of free-text crash narratives. RESULTS: The structured crash data in each state include fields that allowed farm vehicles or equipment and logging trucks to be identified. The variable definitions and coding were not consistent across states but could be harmonized. All states recorded data fields pertaining to person, vehicle, and crash/environmental factors. Structured data supported the construction of crash severity models and geospatial analyses. Law enforcement provided additional details on crash causation in free-text narratives. Crash narratives contained sufficient text to support viable machine learning models for farm vehicle or equipment crashes, but not for logging truck narratives. DISCUSSION: Crash records can help to fill research and surveillance gaps in AgFF in the southwest region. This supports traffic safety's evolution to the current Safe System paradigm. There is a conceptual linkage between the Safe System and Total Worker Health approaches, providing a bridge between traffic safety and occupational health. PRACTICAL APPLICATIONS: Despite limitations, crash records can be an important component of injury surveillance for events involving AgFF vehicles. They also can be used to inform the selection and evaluation of traffic countermeasures and behavioral interventions.

Investigating the impact of environmental and temporal features on mobile phone distracted driving behavior using phone use data.

Mobile phone distracted driving (MPDD) is one of the most significant and common factors in distraction-affected crashes. In previous studies, MPDD has been described as a self-selected behavior that affects driving performance, rather than a multidimensionally impacted behavior. In this study, the researchers hypothesized that external environmental features significantly impacted MPDD and tested this hypothesis by structural equation modeling (SEM). Three external latent variables (road, operation, and control factors) were measured at different times during weekdays in urban areas of Texas by integrating a large number of mobile phone sensor data and roadway inventory data. A structural model was developed to test the relationship between the latent variables and the rate of drivers involved in MPDD (MPDDR) on the roadway during different time periods. Finally, the data summary and model results revealed significant temporal effects. Standardized estimates from the SEM results revealed the positive impact of roads factors in the morning peak that broader shoulders, wider medians, and smaller curve radians were correlated with higher MPDDR in the morning peak hours; the negative impact of operation factors that higher average annual daily truck traffic (truck AADT) were associated with lower MPDDR significantly. And the impact of control factors on MPDDR is positive. In other words, the road segments with a large number of traffic signals in urban areas had a higher MPDDR than those without traffic signals. These findings could assist transportation and legislation agencies in the development of appropriate countermeasures or enforcement tactics and implement them effectively to reduce the occurrence of MPDD. In addition, this study provides a novel perspective close to the actual consideration of drivers about using mobile phones while driving, in the context of MPDD research, rather than comparing driver groups and vehicle performance.

Inclusion of phone use while driving data in predicting distraction-affected crashes.

INTRODUCTION: Given the tremendous number of lives lost or injured, distracted driving is an important safety area to study. With the widespread use of cellphones, phone use while driving has become the most common distracted driving behavior. Although researchers have developed safety performance functions (SPFs) for various crash types, SPFs for distraction-affected crashes are rarely studied in the literature. One possible reason is the lack of critical distracted behavior information in the commonly used safety data (i.e., roadway inventory, traffic, and crash counts). Recently, the frequency of phone use while driving (referred to as phone use data) is recorded by mobile application companies and has become available to safety researchers. The primary objective of this study is to examine if phone use data can potentially predict distracted-affected crashes. METHOD: The authors first integrated phone use data with roadway inventory, traffic, and crash data in Texas. Then, the Random Forest (RF) algorithm was applied to assess the significance of the feature - phone use while driving - for predicting the number of distraction-affected crashes on a road segment. Further, this study developed two SPFs for distraction-affected crashes with and without the phone use data, separately. Both SPFs were assessed in terms of model fitting and prediction performances. RESULTS: RF results rank the frequency of phone use as an important factor contributing to the number of distraction-affected crashes. Performance evaluations indicated that the inclusion of phone use data in the SPFs consistently improved both fitting and prediction abilities to predict distracted-affected crashes. Practical Applications: The phone use data provide new insights into the safety analyses of distraction-affected crashes, which cannot be achieved by only using the conventional roadway inventory and crash data. Therefore, safety researchers and practitioners are encouraged to incorporate the emerging data sources in reducing distraction-affected crashes.

Quantifying and comparing the effects of key risk factors on various types of roadway segment crashes with LightGBM and SHAP.

Understanding and quantifying the effects of risk factors on crash frequency is of great importance for developing cost-effective safety countermeasures. In this paper, the effects of key crash contributing factors on total crashes and crashes of different collision types are analyzed separately and compared. A novel Machine Learning (ML) method, Light Gradient Boosting Machine (LightGBM), is introduced to model a Texas dataset consisting of vehicle crashes occurred from 2015 to 2017. Compared with other commonly used ML methods such as eXtreme Gradient Boosting (XGBoost), LightGBM performs significantly better in terms of mean absolute error (MAE) and root mean squared error (RMSE). In addition, the SHapley Additive explanation (SHAP) approach is employed to interpret the LightGBM outputs. Significant risk factors are identified, including speed limits, area type, number of lanes, roadway functional class, shoulder width and shoulder type. With the SHAP method, the importance, total effects, and main and interaction effects of risk factors are quantified. The results suggest that the importance of risk factors vary across collision types. Speed limit is a more important risk factor than right/left shoulder width, lane width, and median width for Rear-End (RE) crashes, while the opposite relationship is found for Run-Off-Road (ROR) crashes. Also, it is found that narrow lanes (8ft to 11ft) increase the risk for all types of crashes (i.e., Total, ROR, and RE) in this study. For road segments with 5 or 6 lanes in both directions combined, a lane width greater than or equal to 12ft may help reduce the risk of all types of crashes. These results have important implications for developing accurate crash modification factors and cost-effective safety countermeasures.

Generalized criteria for evaluating hotspot identification methods.

Hotspot identification (HSID) is one of the most important components in the highway safety management process. Previous research has found that hazardous sites identified with different methods are not consistent. It is therefore necessary to evaluate the performance of various HSID methods. The existing evaluation criteria are limited to two consecutive periods, and do not consider the temporal instability of crashes. In addition, one existing criterion does not precisely evaluate HSID method under given circumstances. This paper proposed three generalized criteria to evaluate the performance of HSID methods: (1) High Crashes Consistency Test (HCCT) is proposed to evaluate HSID methods in terms of their reliabilities of identifying sites with high crash counts; (2) Common Sites Consistency Test (CSCT) is proposed to gauge HSID methods in consistently identifying a set of common sites as hazardous sites; and, (3) Absolute Rank Differences Test (ARDT) is proposed to measure the consistency of HSID methods in measuring the absolute differences in rankings. Further, three commonly used HSID methods are applied to estimate crashes on Texas rural two-lane roadway segments with eight years of crash data. The performance of these three HSID methods were evaluated to validate the proposed criteria. Comparisons between the existing criteria and the generalized criteria revealed that: (1) the generalized criteria are capable of evaluating different HSID methods over multiple periods; and (2) the generalized criteria are enhanced with a consistent result and with less discrepancy in scores of the best identified HSID method.

Understanding crash potential associated with teen driving: Survey analysis using multivariate graphical method.

INTRODUCTION: Teen crash involvement is usually higher than other age groups, and they are typically overrepresented in car crashes. To infer teen drivers' understanding of crash potentials (factors that are associated with crash occurrence), two sources of data are generally used: retrospective data and prospective data. Retrospective data sources contain historical crash data, which have limitations in determining teen drivers' knowledge of crash potentials. Prospective data sources, like surveys, have more potential to minimize the research gap. Prior studies have shown that teen drivers are more likely to be involved in crashes during their early driving years. Thus, there is a benefit in examining how teen drivers' understanding of crash potentials change during their transition through licensing stages (i.e., no licensure to unrestricted licensure). METHOD: This study used a large set of teen driver survey data (a dataset from approximately 88,000 respondents) of Texas teens to answer the research question. Researchers provided rankings of the crash potentials by gender and licensure stages using a multivariate graphical method named taxicab correspondence analysis (TCA). RESULTS: The findings show that driving behavior and understanding of crash potentials differ among teens based upon various licensing stages. Practical applications: Findings from this study can help government authorities to refine policies of teen driver licensing and implement potential countermeasures for safety improvement.

[Acupoint application of herbal paste relieves symptoms and naso-mucosal inflammatory response by down-regulating serum TGF-ß1 in allergic rhinitis rats].

OBJECTIVE: To observe the effect of acupoint application of herbal paste on symptoms of allergic rhinitis (AR), serum immunoglobulin E (IgE) and transforming growth factor beta 1 (TGF-ß1) level, and number of nasal eosinophils (EOS) in rats with AR, so as to explore its underlying mechanisms. METHODS: Forty male Wistar rats were randomly divided into normal control, model, medication and acupoint application groups (n＝10 rats per group). The AR model was established by intraperitoneal (i.p.) injection of mixture solution of ovalbumin, aluminum hydroxide and normal saline (once every other day, for 7 times), and nasal drip plus spray inhalation of ovalbumin (on the following day of i．p., once daily for 9 days). For acupoint application, the prepared herbal paste (containing White Mustard Seed, Rhizoma Corydalis, unprocessed Radix Kansui, Herba Asari and ginger juice) was applied to bilateral "Feishu" (BL13), "Pishu" (BL20) and "Shenshu" (BL23) for 2 h, once every other day for 7 times. The rats in the medication group were given Fluticasone Propionate nasal spray daily for 14 days. Scores of nasal itching, sneezing and nasal discharge on the day after modeling and the ending of the intervention were used to evaluate behavioral changes. Enzyme linked immunosorbent assay (ELISA) was used to detect the levels of serum IgE and TGF-ß1, and the infiltration state of EOS in the nasal mucosa tissue was observed under light microscope after HE staining. RESULTS: After modeling and compared with the normal control group, the behavioral scores and the levels of serum IgE and TGF-ß1 were significantly higher (P<0.05), and the infiltration state of EOS got worse. Compared with the model group, the increased behavioral score and serum IgE and TGF-ß1 levels were evidently suppressed (P<0.05) and EOS infiltration severity in the nasal mucosa was obviously milder in both medication and acupoint application groups. No significant differences were found between the medication and acupoint application groups in behavioral score and serum IgE and TGF-ß1 levels (P>0.05). CONCLUSION: Acupoint application can improve the symptoms of AR rats, which may be associated with its effect in down-regulating the levels of serum IgE and TGF-ß1.

Strategies to generate functionally normal neutrophils to reduce infection and infection-related mortality in cancer chemotherapy.

Neutrophils form an essential part of innate immunity against infection. Cancer chemotherapy-induced neutropenia (CCIN) is a condition in which the number of neutrophils in a patient's bloodstream is decreased, leading to increased susceptibility to infection. Granulocyte colony-stimulating factor (GCSF) has been the only approved treatment for CCIN over two decades. To date, CCIN-related infection and mortality remain a significant concern, as neutrophils generated in response to administered GCSF are functionally immature and cannot effectively fight infection. This review summarizes the molecular regulatory mechanisms of neutrophil granulocytic differentiation and innate immunity development, dissects the biology of GCSF in myeloid expansion, highlights the shortcomings of GCSF in CCIN treatment, updates the recent advance of a selective retinoid agonist that promotes neutrophil granulocytic differentiation, and evaluates the benefits of developing GCSF biosimilars to increase access to GCSF biologics versus seeking a new mode to fundamentally advance GCSF therapy for treatment of CCIN.

In Vivo Study on Site of Action of Sinapine Thiocyanate following Acupoint Herbal Patching.

OBJECTIVE: To investigate the site of action of sinapine thiocyanate (ST), following acupoint herbal patching (AHP). METHODS: Twenty Wistar rats were randomized into five groups (groups A, B, C, D, and E), and all groups received the same AHP in vivo. Skin samples were excised at 2 h, 4 h, 6 h, 10 h, and 26 h after AHP administration from group A to group E separately and the concentrations of ST in the skin were determined using a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method. A pharmacokinetic profile of ST following AHP was performed at the same time in a group of five Wistar rats to detect plasma levels at the same time intervals. RESULTS: The mean ± SD ST concentrations (ng/ml) at 2 h (group A), 4 h (group B), 6 h (group C), 10 h (group D), and 26 h (group E) after AHP administration were 250.01 ± 61.99, 61.01 ± 30.41, 40.12 ± 26.94, 78.66 ± 59.43, and 19.55 ± 18.95, respectively. No ST was detected in rats' plasma samples at the same time points. CONCLUSIONS: The site of action of ST following AHP is in the skin.

Examining the influence of link function misspecification in conventional regression models for developing crash modification factors.

This study further examined the use of regression models for developing crash modification factors (CMFs), specifically focusing on the misspecification in the link function. The primary objectives were to validate the accuracy of CMFs derived from the commonly used regression models (i.e., generalized linear models or GLMs with additive linear link functions) when some of the variables have nonlinear relationships and quantify the amount of bias as a function of the nonlinearity. Using the concept of artificial realistic data, various linear and nonlinear crash modification functions (CM-Functions) were assumed for three variables. Crash counts were randomly generated based on these CM-Functions. CMFs were then derived from regression models for three different scenarios. The results were compared with the assumed true values. The main findings are summarized as follows: (1) when some variables have nonlinear relationships with crash risk, the CMFs for these variables derived from the commonly used GLMs are all biased, especially around areas away from the baseline conditions (e.g., boundary areas); (2) with the increase in nonlinearity (i.e., nonlinear relationship becomes stronger), the bias becomes more significant; (3) the quality of CMFs for other variables having linear relationships can be influenced when mixed with those having nonlinear relationships, but the accuracy may still be acceptable; and (4) the misuse of the link function for one or more variables can also lead to biased estimates for other parameters. This study raised the importance of the link function when using regression models for developing CMFs.

Finite mixture modeling approach for developing crash modification factors in highway safety analysis.

This study aimed to investigate the relative performance of two models (negative binomial (NB) model and two-component finite mixture of negative binomial models (FMNB-2)) in terms of developing crash modification factors (CMFs). Crash data on rural multilane divided highways in California and Texas were modeled with the two models, and crash modification functions (CMFunctions) were derived. The resultant CMFunction estimated from the FMNB-2 model showed several good properties over that from the NB model. First, the safety effect of a covariate was better reflected by the CMFunction developed using the FMNB-2 model, since the model takes into account the differential responsiveness of crash frequency to the covariate. Second, the CMFunction derived from the FMNB-2 model is able to capture nonlinear relationships between covariate and safety. Finally, following the same concept as those for NB models, the combined CMFs of multiple treatments were estimated using the FMNB-2 model. The results indicated that they are not the simple multiplicative of single ones (i.e., their safety effects are not independent under FMNB-2 models). Adjustment Factors (AFs) were then developed. It is revealed that current Highway Safety Manual's method could over- or under-estimate the combined CMFs under particular combination of covariates. Safety analysts are encouraged to consider using the FMNB-2 models for developing CMFs and AFs.

Am80-GCSF synergizes myeloid expansion and differentiation to generate functional neutrophils that reduce neutropenia-associated infection and mortality.

Neutrophils generated by granulocyte colony-stimulating factor (GCSF) are functionally immature and, consequently, cannot effectively reduce infection and infection-related mortality in cancer chemotherapy-induced neutropenia (CCIN). Am80, a retinoic acid (RA) agonist that enhances granulocytic differentiation by selectively activating transcription factor RA receptor alpha (RARα), alternatively promotes RA-target gene expression. We found that in normal and malignant primary human hematopoietic specimens, Am80-GCSF combination coordinated proliferation with differentiation to develop complement receptor-3 (CR3)-dependent neutrophil innate immunity, through altering transcription of RA-target genes RARß2, C/EBPε, CD66, CD11b, and CD18 This led to generation of functional neutrophils capable of fighting infection, whereas neutralizing neutrophil innate immunity with anti-CD18 antibody abolished neutrophil bactericidal activities induced by Am80-GCSF Further, Am80-GCSF synergy was evaluated using six different dose-schedule-infection mouse CCIN models. The data demonstrated that during "emergency" granulopoiesis in CCIN mice undergoing transient systemic intravenous bacterial infection, Am80 effect on differentiating granulocytic precursors synergized with GCSF-dependent myeloid expansion, resulting in large amounts of functional neutrophils that reduced infection. Importantly, extensive survival tests covering a full cycle of mouse CCIN with perpetual systemic intravenous bacterial infection proved that without causing myeloid overexpansion, Am80-GCSF generated sufficient numbers of functional neutrophils that significantly reduced infection-related mortality in CCIN mice. These findings reveal a differential mechanism for generating functional neutrophils to reduce CCIN-associated infection and mortality, providing a rationale for future therapeutic approaches.

The lost intrinsic fragmentation of MAT1 protein during granulopoiesis promotes the growth and metastasis of leukemic myeloblasts.

MAT1, an assembly factor and targeting subunit of both cyclin-dependent kinase-activating kinase (CAK) and general transcription factor IIH (TFIIH) kinase, regulates cell cycle and transcription. Previous studies show that expression of intact MAT1 protein is associated with expansion of human hematopoietic stem cells (HSC), whereas intrinsically programmed or retinoic acid (RA)-induced MAT1 fragmentation accompanies granulocytic differentiation of HSC or leukemic myeloblasts. Here we determined that, in humanized mouse microenvironment, MAT1 overexpression resisted intrinsic MAT1 fragmentation to sustain hematopoietic CD34+ cell expansion while preventing granulopoiesis. Conversely, we mimicked MAT1 fragmentation in vitro and in a mouse model by overexpressing a fragmented 81-aa MAT1 polypeptide (pM9) that retains the domain for assembling CAK but cannot affix CAK to TFIIH-core. Our results showed that pM9 formed ΔCAK by competing with MAT1 for CAK assembly to mimic MAT1 fragmentation-depletion of CAK. This resulting ΔCAK acted as a dominant negative to inhibit the growth and metastasis of different leukemic myeloblasts, with or without RA resistance, by concurrently suppressing CAK and TFIIH kinase activities to inhibit cell cycle and gene transcription. These findings suggest that the intrinsically programmed MAT1 expression and fragmentation regulate granulopoiesis by inversely coordinating CAK and TFIIH activities, whereas pM9 shares a mechanistic resemblance with MAT1 fragmentation in suppressing myeloid leukemogenesis.

Retinoid agonist Am80-enhanced neutrophil bactericidal activity arising from granulopoiesis in vitro and in a neutropenic mouse model.

Despite advances in the therapeutic use of recombinant granulocyte colony-stimulating factor (G-CSF) to promote granulopoiesis of human hematopoietic stem cells (HSCs), neutropenia remains one of the most serious complications of cancer chemotherapy. We discovered that retinoid agonist Am80 (tamibarotene) is more potent than G-CSF in coordinating neutrophil differentiation and immunity development. Am80-induced neutrophils (AINs) either in vitro or in neutropenic mouse model displayed strong bactericidal activities, similar to those of human peripheral blood neutrophils (PBNs) or mouse peripheral blood neutrophils (MPBNs) but markedly greater than did G-CSF­induced neutrophils (GINs). In contrast to GINs but similar to PBNs, the enhanced bacterial killing by AINs accompanied both better granule maturation and greater coexpression of CD66 antigen with the integrin ß2 subunit CD18. Consistently, anti-CD18 antibody neutralized Am80-induced bactericidal activities of AINs. These studies demonstrate that Am80 is more effective than G-CSF in promoting neutrophil differentiation and bactericidal activities, probably through coordinating the functional interaction of CD66 with CD18 to enhance the development of neutrophil immunity during granulopoiesis. Our findings herein suggest a molecular rationale for developing new therapy against neutropenia using Am80 as a cost-effective treatment option.

Retinoid-regulated FGF8f secretion by osteoblasts bypasses retinoid stimuli to mediate granulocytic differentiation of myeloid leukemia cells.

Signaling from the human hematopoietic stem cell (HSC) niche formed by osteoblastic cells regulates hematopoiesis. We previously found that retinoic acid receptor alpha (RARα), a transcription factor activated by retinoic acid (RA), mediates both granulocytic and osteoblastic differentiation. This effect depends on decreased phosphorylation of serine 77 of RARα (RARαS77) by the cyclin-dependent kinase-activating kinase (CAK) complex, a key cell-cycle regulator. In this article, we report that, by suppressing CAK phosphorylation of RARα, RA induces FGF8f to mediate osteosarcoma U2OS cell differentiation in an autocrine manner. By contrast, paracrine FGF8f secreted into osteoblast-conditioned medium by U2OS cells transduced with FGF8f or a phosphorylation-defective RARαS77 mutant, RARαS77A, bypasses RA stimuli to cross-mediate granulocytic differentiation of different types of human leukemic myeloblasts and normal primitive hematopoietic CD34(+) cells, possibly through modulating mitogen-activated protein kinase (MAPK) pathways. Further experiments using recombinant human FGF8f (rFGF8f) stimuli, antibody neutralization, and peptide blocking showed that paracrine FGF8f is required for mediating terminal leukemic myeloblast differentiation. These studies indicate a novel regulatory mechanism of granulocytic differentiation instigated by RA from the HSC niche, which links loss of CAK phosphorylation of RARα with paracrine FGF8f-mediated MAPK signaling to mediate leukemic myeloblast differentiation in the absence of RA. Therefore, these findings provide a compelling molecular rationale for further investigation of paracrine FGF8f regulation, with the intent of devising HSC niche-based FGF8f therapeutics for myeloid leukemia, with or without RA-resistance.

Loss of CAK phosphorylation of RAR{alpha} mediates transcriptional control of retinoid-induced cancer cell differentiation.

Although the role of the classic retinoic acid (RA)-induced genomic pathway in cancer cell differentiation is well recognized, the underlying mechanisms remain to be dissected. Retinoic acid receptor alpha (RARalpha) is a transcription factor activated by RA, and its serine 77 (RARalphaS77) is the main residue phosphorylated by the cyclin-dependent kinase (CDK)-activating kinase (CAK) complex. We report here that in both human myeloid leukemia and mouse embryonic teratocarcinoma stem cells, either RA-suppressed CAK phosphorylation of RARalpha or mutation of RARalphaS77 to alanine (RARalphaS77A) coordinates CAK-dependent G(1) arrest with cancer cell differentiation by transactivating RA-target genes. Both hypophosphorylated RARalpha and RARalphaS77A reduce binding to retinoic acid-responsive elements (RARE) in the promoters of RA-target genes while stimulating gene transcription. The enhanced transactivation and reduced RARalpha-chromatin interaction are accompanied by RARalpha dissociation from the transcriptional repressor N-CoR and are association with the coactivator NCoA-3. Such effects of decreased CAK phosphorylation of RARalphaS77 on mediating RA-dependent transcriptional control of cancer cell differentiation are examined correspondingly in both RA-resistant myeloid leukemia and embryonic teratocarcinoma stem RARalpha(-/-) cells. These studies demonstrate, for the first time, that RA couples G(1) arrest to transcriptional control of cancer cell differentiation by suppressing CAK phosphorylation of RARalpha to release transcriptional repression.-Wang, A., Alimova, I. N., Luo, P. Jong, A., Triche, T. J., Wu, L. Loss of CAK phosphorylation of RARalpha mediates transcriptional control of retinoid-induced cancer cell differentiation.

Cdc6 knockdown inhibits human neuroblastoma cell proliferation.

The cell division controller Cdc6 plays a central role in the initiation of DNA replication. It was found that elevated levels of Cdc6 were present in many of human cancer cells, and the accumulation of Cdc6 is required for cell proliferation. In this study, we have investigated the control of Cdc6 expression and its effect on cell proliferation and death in human neuroblastoma cells. Elevated levels of Cdc6 are found in the LA-N-2, CHLA255, and other cell lines that grow fast. Cdc6 knockdown via a Cdc6 short hairpin RNA lentivirus causes the accumulation of sub-G1 populations with the decrease of S contents in the LA-N-2 and CHLA255 cells. Expression profile from the selected genes shows the reduction of cyclin E, cyclin A, and Cdc25C, with a boosted increase of the CDK inhibitor p27Kip1, indicating the suppression of tumor cell proliferation. Further, Cdc6 knockdown causes the increase of pro-apoptotic Bax accompanied with the decrease of anti-apoptotic Bcl-2, resulting in the increased cell death. Furthermore, Cdc6 knockdown causes a sharp reduction of tumor suppressor protein p53, and Cdc6 overexpression renders a boosted p53 expression; and this regulation is at p53 posttranscriptional level. Our study indicates that human Cdc6 functions in several pathways to control the cell proliferation and the cell death.

Intrinsic retinoic acid receptor alpha-cyclin-dependent kinase-activating kinase signaling involves coordination of the restricted proliferation and granulocytic differentiation of human hematopoietic stem cells.

Little is known about the mechanisms by which retinoic acid receptor alpha (RAR alpha) mediates the effects of retinoic acid (RA) to coordinate granulocytic proliferation/differentiation (P/D) transition. Cyclin-dependent kinase-activating kinase (CAK) complex, whose activity in phosphorylation of RAR alpha is determined by its targeting subunit ménage à trois 1 (MAT1), regulates G(1) exit, a cell cycle stage when cells commonly commit to proliferation or to differentiation. We previously found that in myeloid leukemia cells, the lack of RA-induced RAR alpha-CAK dissociation and MAT1 degradation suppresses cell differentiation by inhibiting CAK-dependent G(1) exit and sustaining CAK hyperphosphorylation of RAR alpha. This contrasts with our recent findings about the P/D transition in normal primitive hematopoietic cells, where MAT1 degradation proceeds intrinsically together with granulocytic development, in accord with dynamic expression of aldehyde dehydrogenases (ALDHs) 1A1 and 1B1, which catalyze RA synthesis. Blocking ALDH activity inhibits MAT1 degradation and granulocytic differentiation, whereas loss of RAR alpha phosphorylation by CAK induces RA-target gene expression and granulocytic differentiation. These studies suggest that the subversion of RAR alpha-CAK signaling during normal granulopoiesis is crucial to myeloid leukemogenesis and challenges the current paradigm that RA induces cell differentiation solely by transactivating target genes. Disclosure of potential conflicts of interest is found at the end of this article.

Downregulation of human Cdc6 protein using a lentivirus RNA interference expression vector.

Eukaryotic CDC6 gene function is required for the initiation of DNA replication and is a key regulatory protein during cell cycle progression. The human CDC6 gene is not expressed in most normal tissues, in contrast with its marked expression in proliferating cancer cells. An effective way to explore the gene functions of CDC6 is to knock-down the CDC6 messenger RNA (mRNA) and examine the phenotypic consequences. In this chapter, we describe the construction of a lentivirus vector to express a CDC6 DNA segment. The transcript is able to fold by itself because the sense and antisense regions are complementary. There is a 9-nucleotide (nt) loop region allowing for the short hairpin RNA (shRNA) to form. Cellular ribonucleases process the shRNA into a functional short interfering RNA (siRNA). Down-regulation of Cdc6 protein is confirmed by Western blots.

Retinoic acid induces leukemia cell G1 arrest and transition into differentiation by inhibiting cyclin-dependent kinase-activating kinase binding and phosphorylation of PML/RARalpha.

Acute promyelocytic leukemia (APL) cells express promyelocytic leukemia/retinoic acid receptor alpha (PML/RARalpha) fusion protein, which leads to the blocking of APL cell differentiation. Treatment of APL with all-trans-retinoic acid (ATRA) induces disease remission by in vivo differentiation of APL cells. Differentiation requires cell cycle exit; yet how ATRA couples cell cycle exit to differentiation of APL remains largely unknown. We previously found that ATRA-induced cell differentiation accompanies ubiquitination-proteolysis of ménage à trois 1 (MAT1), an assembly factor and targeting subunit of cyclin-dependent kinase (CDK)-activating kinase (CAK) that regulates G1 exit. We report here that CAK binds to and phosphorylates PML/RARalpha in actively proliferating APL cells. In response to ATRA, PML/RARalpha is dissociated from CAK, leading to MAT1 degradation, G1 arrest, and decreased CAK phosphorylation of PML/RARalpha. CAK phosphorylation of PML/RARalpha is inhibited when MAT1 levels are reduced. Both MAT1 degradation and PML/RARalpha hypophosphorylation occur in ATRA-induced G1-arresting cells undergoing differentiation but not in the synchronized G1 cells that do not differentiate. These findings reveal a novel ATRA signaling on APL cell differentiation, in which ATRA coordinates G1 arrest and transition into differentiation by inducing MAT1 degradation and PML/RARalpha hypophosphorylation through disrupting PML/RARalpha binding and phosphorylation by CAK.