[A Case of Xanthogranulomatous Pyelonephritis with Multiple Lymphadenopathy that was Difficult to Differentiate from Renal Tumor].

A 74-year-old woman presented to our hospital with the main complaint of anorexia and weight loss for several months. Computed tomography (CT) revealed right urinary stone, hydronephrosis, multiple lymphadenopathy, and a mass in the right kidney. Considering these findings, she was suspected to have renal malignancy (kidney or renal pelvis cancer) with multiple lymph node metastases; therefore, nephrectomy was performed. Her pathological diagnosis was xanthogranulomatous pyelonephritis (XGPN). There was no postoperative renal function decline, and multiple lymphadenopathy also disappeared on CT 3 months after surgery. It was judged to be reactive swelling due to inflammation. XGPN is a pathological condition characterized by accumulation of mast cells and activated macrophages in the renal tissue; and, the renal tissue recognizes yellowish granulation growth because of repeating pyelonephritis due to urinary tract passing impairment. In some cases, it is difficult to differentiate XGPN from renal malignancy. Moreover, lymphadenopathy may be lymph node metastasis but may also present reactive enlargement due to the effect of inflammation, making it even more difficult to differentiate when accompanied by lymphadenopathy. We report this case in which it was difficult to differentiate XGPN from renal malignancy considering the scarcity of reports of XGPN accompanied by multiple lymphadenopathy.

Successful treatment of eosinophilia associated with dialysis-related renal cancer with radical nephrectomy.

Introduction: Secondary eosinophilia due to solid tumors is a rare case. This is the first study to report secondary eosinophilia due to renal cancer in a patient on dialysis. Case presentation: A 70-year-old man, on long-term hemodialysis was incidentally detected with right renal cancer, and workup performed revealed eosinophilia. Allergic symptoms caused by hemodialysis were initially considered; however, treatment did not lead to any improvement in eosinophilia. Therefore, nephrectomy for renal cancer was performed. The resolution of symptoms and eosinophilia after surgery suggested renal cancer as the cause of eosinophilia. Conclusion: As demonstrated in this patient with dialysis-related renal cancer, eosinophilia associated with solid tumors may be addressed by treating the tumor.

Prospective Registration Study for Establishing Minimal Clinically Important Differences in Patients Undergoing Surgery for Spinal Metastases.

STUDY DESIGN: Multicenter, prospective registry study. OBJECTIVE: To clarify minimal clinically important differences (MCIDs) for surgical interventions for spinal metastases, thereby enhancing patient care by integrating quality of life (QoL) assessments with clinical outcomes. SUMMARY OF BACKGROUND DATA: Despite its proven usefulness in degenerative spinal diseases and deformities, the MCID remains unexplored regarding surgery for spinal metastases. METHODS: This study included 171 (out of 413) patients from the multicenter "Prospective Registration Study on Surgery for Metastatic Spinal Tumors" by the Japan Association of Spine Surgeons. These were evaluated preoperatively and at 6 months postoperatively using the Face scale, EuroQol-5 Dimensions-5 Levels (EQ-5D-5L), including the visual analog scale (VAS), and performance status. The MCIDs were calculated using an anchor-based method, classifying participants into the improved, unchanged, and deteriorated groups based on the Face scale scores. Focusing on the improved and unchanged groups, the change in the EQ-5D-5L values from before to after treatment was analyzed, and the cutoff value with the highest sensitivity and specificity was determined as the MCID through receiver operating characteristic curve analysis. The validity of the MCIDs was evaluated using a distribution-based calculation method for patient-reported outcomes. RESULTS: The improved, unchanged, and deteriorated groups comprised 121, 28, and 22 participants, respectively. The anchor-based MCIDs for the EQ-5D-5L index, EQ-VAS, and domains of mobility, self-care, usual activities, pain/discomfort, and anxiety/depression were 0.21, 15.50, 1.50, 0.50, 0.50, 0.50, and 0.50, respectively; the corresponding distribution-based MCIDs were 0.17, 15,99, 0.77, 0.80, 0.78, 0.60, and 0.70, respectively. CONCLUSION: We identified MCIDs for surgical treatment of spinal metastases, providing benchmarks for future clinical research. By retrospectively examining whether the MCIDs are achieved, factors favoring their achievement and risks affecting them can be explored. This could aid in decisions on surgical candidacy and patient counseling.

Age and parous-experience dependent changes in emotional contagion for positive infant sounds.

Introduction: Emotional contagion is achieved by inferring and emotionally resonating with other persons' feelings. It is unclear whether age-related changes in emotional contagion for infant sounds are modulated by the experience of childbirth or childcare. This study aims to evaluate changes in inference and emotional resonance for positive and negative infant sounds (laughter and crying) among women, based on age and parous experience. Methods: A total of 241 women (60 young nulliparous, 60 young parous, 60 old nulliparous, and 61 old parous) completed a web-based questionnaire. After listening to three types of infant sounds (laughter, cooing, and crying), participants responded with their valence for hearing infant sounds and estimated infant valence on an 11-point Likert scale. Results: The analysis for emotional resonance revealed that the correlation coefficient between self and estimated infant valences was greater in young parous and old nulliparous women than in young nulliparous women, in laughter and cooing sounds. However, correlation coefficients for crying did not differ among any of the four groups. Conclusion: The degree of emotional resonance for infant valence increased depending on age and parous-experience for positive infant sounds.

A case of complete response to avelumab plus axitinib combination therapy for metastatic clear cell renal cell carcinoma in a kidney undergoing dialysis.

Introduction: Combination therapies of immune checkpoint and tyrosine kinase inhibitors for end-stage kidney disease and patients on hemodialysis need careful consideration as few case reports provide suitable management decisions. Case presentation: A 70-year-old man who had undergone hemodialysis for 6 years due to nephrosclerosis. Avelumab plus axitinib combination therapy was performed for repeated lung metastasis, and a complete response was achieved without major side effects. Conclusion: A complete response was achieved after Ave plus Axi combination therapy for clear cell renal cell carcinoma in a patient undergoing dialysis. This suggests that Ave plus Axi combination therapy may be safe and effective for dialysis patients.

Transforming Growth Factor-ß Induces Interleukin-6 Secretion from Human Ligamentum Flavum-Derived Cells through Partial Activation of p38 and p44/42 Mitogen-Activated Protein Kinases.

STUDY DESIGN: This experimental study was performed using human ligamentum flavum-derived cells (HFCs). PURPOSE: To investigate the intracellular signaling mechanism of interleukin-6 (IL-6) secretion in transforming growth factor-ß (TGF- ß)-stimulated HFCs. OVERVIEW OF LITERATURE: Lumbar spinal stenosis (LSS) is a prevalent disease among the elderly, characterized by debilitating pain in the lower extremities. Although the number of patients with LSS has increased in recent years, the underlying pathomechanism remains unclear. Clinical examinations typically rely on magnetic resonance imaging to diagnose patients, revealing ligamentum flavum hypertrophy. Some studies have suggested an association between ligamentum flavum hypertrophy and inflammation/fibrosis, and expression of TGF-ß and IL-6 has been observed in surgically obtained ligamentum flavum samples. However, direct evidence linking TGF-ß and IL-6 expression in HFCs is lacking. METHODS: HFCs were obtained from patients with LSS who had undergone decompression surgery. The cells were stimulated with TGF-ß and pretreated with either the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 or the p44/42 MAP kinase inhibitor FR180204. IL-6 secretion in the cell culture medium and IL-6 messenger RNA (mRNA) expression levels were analyzed using an enzyme-linked immunoassay and real-time polymerase chain reaction, respectively. RESULTS: TGF-ß administration resulted in a dose- and time-dependent stimulation of IL-6 release. Treatment with SB203580 and FR180204 markedly suppressed TGF-ß-induced IL-6 secretion from HFCs. Moreover, these inhibitors suppressed IL-6 mRNA expression in response to TGF-ß stimulation. CONCLUSIONS: Our findings indicate that TGF-ß induces IL-6 protein secretion and gene expression in HFCs through the activation of p38 or p44/42 MAP kinases. These results suggest a potential association between IL-6-mediated inflammatory response and tissue hypertrophy in LSS, and we provide insights into molecular targets for therapeutic interventions targeting LSS-related inflammation through our analysis of the MAP kinase pathway using HFCs.

Interlimb and Intralimb Coordination of Rectus Femoris and Biceps Femoris Muscles at Different Running Speeds.

PURPOSE: The purpose of this study was to investigate the relationship between spatiotemporal variables and the muscle activity of the rectus femoris (RF) and biceps femoris (BF) in both legs at various running speeds. METHODS: Eighteen well-trained male athletes (age: 20.7 ± 1.8 yr) were asked to run for 50 m with 7 different "subjective efforts (SE)" (20%, 40%, 60%, 80%, 90%, 95%, and 100% SE). SE scaled relative to the maximal effort running (100%). The spatiotemporal variables (running speed, step frequency, step length) were measured over the distance from 30 to 50 m. The RF and BF muscle activities were obtained from both legs with wireless electromyography (EMG) sensors. We calculated RF and BF onset/offset timings in both legs (e.g., ipsilateral leg RF is "iRF," contralateral leg BF is "cBF"), which were expressed as % of a running cycle. Based on those timings, we obtained the EMG timing variables (%), as Switch1 (iBF offset to iRF onset), Switch2 (iRF offset to iBF onset), Scissors1 (cBF onset to iRF onset), and Scissors2 (iRF offset to cBF offset). RESULTS: running speed was well correlated with the SE, and higher running speed (>9 m·s -1 ) was achieved with higher step frequency (>4.0 Hz). Relative timings of RF and BF onset/offset (%) appeared earlier and later, respectively, with an increase in running speed. The absolute duration of RF activation (s) was elongated with the decrease in absolute running cycle time (increase in running speed). Both Switch and Scissors showed significant negative correlations with running speed and step frequency. CONCLUSIONS: The RF and BF excitation in both legs, as evidenced by changes in both Switch and Scissors, is coordinated for controlling running speed, as well as step frequency.

The Timing of Thigh Muscle Activity Is a Factor Limiting Performance in the Deceleration Phase of the 100-m Dash.

PURPOSE: We aimed to examine the timing of electromyography activity of the rectus femoris (RF) and biceps femoris (BF) in both legs, as well as spatiotemporal variables (running speed (RS), step frequency (SF), step length (SL)) between the maximal speed (Max) phase (50-70 m) and the deceleration (Dec) phase (80-100 m) of the 100-m dash. METHODS: Nine track and field athletes performed the 100-m dash with maximal effort. Spatiotemporal variables of each 10-m section were measured. A portable wireless data logger was attached to the subject's lower back to record electromyographies. We calculated onset/offset timing (%) of RF and BF in both legs using a Teager-Kaiser Energy Operator filter (e.g., ipsilateral leg RF onset is "iRF-onset," contralateral leg BF onset is "cBF-onset") in a running cycle. RESULTS: The decreased RS in the Dec phase (P < 0.001) was due to a decreased SF (P < 0.001). Moreover, iRF-onset (P = 0.002), iRF-offset (P = 0.008), iBF-offset (P = 0.049), and cBF-offset (P = 0.017) in the Dec phase lagged in the running cycle as compared with the Max phase. Furthermore, the time difference between the swing leg RF activity (iRF-onset) and the contact leg BF activity (cBF-onset; "Scissors1") became bigger in the Dec phase (P = 0.041). Significant negative correlations were found between ΔiRF-onset and ΔSF (P = 0.045), and between ΔiBF-offset and ΔSF (P = 0.036). CONCLUSIONS: The decreased RS and SF in the Dec phase of the 100-m dash would be the delayed timing of the RF and BF activities in the same leg as well as the disturbed interleg muscular coordination.

Segmental Arteries and Veins at Higher Lumbar Levels Can Intersect the Adjacent Caudal Intervertebral Disc in the Anterior Part of the Spinal Column: A Cadaveric Analysis.

STUDY DESIGN: A cadaveric study. PURPOSE: To investigate the anatomical features of segmental arteries and veins in the anterior part of the spinal column to prevent segmental vessel injury. OVERVIEW OF LITERATURE: The lateral transpsoas approach to the lumbar intervertebral discs (IVD) is associated with the risk of segmental vessel injury. Previous studies have described the vascular anatomy on the lateral part of the vertebral body. However, there are no studies that describe the segmental vessels on its anterior aspect. Here, we report the important anatomical features of the segmental arteries and veins that can intersect the anterior part of the IVD. These vessels are considered at risk of vascular injury when placing the anterior retractors during lateral lumbar interbody fusion or cutting the anterior longitudinal ligament during anterior column realignment. METHODS: Five formalin-embalmed human cadavers were used. We assessed the proportion of segmental arteries and veins that intersected the IVD in the L2-L5 range and their course on the anterior part of the spinal column. RESULTS: The segmental arteries and veins commonly intersect the anterior part of the IVD (artery, 28.1%; vein, 42.1%). Seven of 10 (70%) segmental arteries at L2 intersected the IVD, but only one artery intersected the IVD at L3 and L4. The proportions of segmental veins that intersected the IVD were 60%, 50%, and 16.7% at L2, L3, and L4, respectively. CONCLUSIONS: The segmental arteries and veins frequently intersect the IVD in the anterior part of the spinal column. Therefore, it is necessary to consider these individual anatomical features to prevent vascular damage during lateral lumbar interbody fusion surgery.

Spatiotemporal inflection points in human running: Effects of training level and athletic modality.

The effect of the different training regimes and histories on the spatiotemporal characteristics of human running was evaluated in four groups of subjects who had different histories of engagement in running-specific training; sprinters, distance runners, active athletes, and sedentary individuals. Subjects ran at a variety of velocities, ranging from slowest to fastest, over 30 trials in a random order. Group averages of maximal running velocities, ranked from fastest to slowest, were: sprinters, distance runners, active athletes, and sedentary individuals. The velocity-cadence-step length (V-C-S) relationship, made by plotting step length against cadence at each velocity tested, was analyzed with the segmented regression method, utilizing two regression lines. In all subject groups, there was a critical velocity, defined as the inflection point, in the relationship. In the velocity ranges below and above the inflection point (slower and faster velocity ranges), velocity was modulated primarily by altering step length and by altering cadence, respectively. This pattern was commonly observed in all four groups, not only in sprinters and distance runners, as has already been reported, but also in active athletes and sedentary individuals. This pattern may reflect an energy saving strategy. When the data from all groups were combined, there were significant correlations between maximal running velocity and both running velocity and step length at the inflection point. In spite of the wide variety of athletic experience of the subjects, as well as their maximum running velocities, the inflection point appeared at a similar cadence (3.0 ± 0.2 steps/s) and at a similar relative velocity (65-70%Vmax). These results imply that the influence of running-specific training on the inflection point is minimal.

Parent and alkylated PAHs profiles in 11 petroleum fuels and lubricants: Application for oil spill accidents in the environment.

Eleven types of petroleum fuels and lubricants including regular gasoline, premium gasoline, jet fuel, kerosene, light oil, bunker A, bunker A-white, bunker A-low sulfur, bunker C, quench oil and lubricant samples were analyzed for parent and alkylated polycyclic aromatic hydrocarbons (PAHs). Naphthalene was the predominant compound in gasolines, jet fuel and kerosene, constituting > 95% of the parent PAHs, whereas dibenzothiophene and other high molecular weight PAHs were predominant in bunker A and bunker C. PAH compositions in petroleum fuels differ because of differences in their refining temperatures and the boiling points of individual PAHs. Principal component analysis classified into four groups of petroleum fuels. Further, oil samples were clearly separated into five groups based on their ratios of select alkyl homologs (C0/(C0+C1) and C4/(C2+C4) naphthalenes): 'gasolines' 'light oil' 'bunker oils' 'kerosene' and 'quench oil'. A wide variety and detailed profiles of PAHs in petroleum fuels and lubricants in this study can be used for baseline data in oil fingerprinting analyses to identify the potential source of oil spill accidents in the environment.

Anti-tumor effect of trametinib in bladder cancer organoid and the underlying mechanism.

Bladder cancer (BC), a main neoplasm of urinary tract, is usually inoperable and unresponsive to chemotherapy. As a novel experimental model for muscle-invasive BC, we previously established a culture method of dog BC organoids. In the present study, the detailed in vitro and in vivo anti-tumor effects of trametinib were investigated by using this model. In each BC organoid strain, epidermal growth factor receptor (EGFR)/ERK signaling was upregulated compared with normal bladder cells. Trametinib even at a low concentration inhibited the cell viability of BC organoids and the activation of ERK through decreasing expression of c-Myc, ELK1, SIK1, and PLA2G4A. Trametinib arrested cell cycle of BC with few apoptosis. Dual treatment of BC organoids with trametinib and YAP inhibitor, verteporfin extremely inhibited the cell viability with apoptosis induction. Moreover, trametinib induced basal to luminal differentiation of BC organoids by upregulating luminal markers and downregulating basal ones. In vivo, trametinib decreased the tumor growth of BC organoids in mice and the xenograft-derived organoids from trametinib-administered mice showed enhanced sensitivity to carboplatin due to MSH2 upregulation. Our data suggested a new strategy of trametinib-YAP inhibitor or trametinib-carboplatin combination as a promising treatment of BC.

Timing of Rectus Femoris and Biceps Femoris Muscle Activities in Both Legs at Maximal Running Speed.

PURPOSE: The purpose of this study was to investigate the relationship between spatiotemporal variables of running and onset/offset timing of rectus femoris (RF) and biceps femoris (BF) muscle activities in both legs. METHODS: Eighteen male well-trained athletes (age = 20.7 ± 1.8 yr) were asked to run 50 m at maximal speed. The spatiotemporal variables (running speed, step frequency, and step length) over the distance from 30 to 50 m were measured. In addition, RF and BF muscle activities were obtained from both legs using wireless EMG sensors. To quantify the onset and offset timing of muscle activity, the band-pass filtered (20-450 Hz) EMG signal was processed using a Teager-Kaiser energy operator filter. We calculated RF and BF onset/offset timings (%) in both legs (e.g., ipsilateral leg RF [iRF] and contralateral leg BF [cBF]) during running cycle. Based on those timings, we obtained the EMG timing variables (%) as follows: "Switch1 (iBF-offset to iRF-onset)," "Switch2 (iRF-offset to iBF-onset)," "Scissors1 (cBF-onset to iRF-onset)," and "Scissors2 (iRF-offset to cBF-offset). RESULTS: We found that "Switch2" had positive (r = 0.495, P = 0.037), "Scissors1" had negative (r = -0.469, P = 0.049), and "Scissors2" had positive (r = 0.574, P = 0.013) correlations with step frequency. However, these variables had no significant correlations with running speed or step length. CONCLUSIONS: These results indicate that higher step frequency would be achieved by smoother switching of the agonist-antagonist muscle activities and earlier iRF activation relative to the cBF activity. To improve sprint performance, athletes and coaches should consider not only muscle activities in one leg but also coordination of muscle activities in both legs.

p38 Mitogen-Activated Protein Kinase Is Involved in Interleukin-6 Secretion from Human Ligamentum Flavum-Derived Cells Stimulated by Tumor Necrosis Factor-α.

STUDY DESIGN: Human ligamentum flavum-derived cells (HFCs) were obtained from surgical samples for a basic experimental study. PURPOSE: We sought to evaluate the inflammatory response of human ligamentum flavum cells to investigate hypertrophic changes occurring in the ligamentum flavum. OVERVIEW OF LITERATURE: Lumbar spinal stenosis (LSS) is a disease commonly observed in the elderly. The number of patients with LSS has increased over time, yet the pathomechanisms of LSS still have not been fully elucidated. One of the clinical features of LSS is hypertrophy of the ligamentum flavum, which results in narrowing of the lumbar spinal canal. Some reports have suggested that ligamentum flavum hypertrophy is associated with inflammation and fibrosis; meanwhile, the p38 mitogen-activated protein (MAP) kinase is involved in the hypertrophy of human ligamentum flavum cells. METHODS: HFCs were obtained from patients with LSS who underwent surgery. HFCs were stimulated by tumor necrosis factor-α (TNF-α) and a p38 MAP kinase inhibitor, SB203580. Phosphorylation of the p38 MAP kinase was analyzed by western blotting. The concentration of interleukin-6 (IL-6) in the conditioned medium was measured by enzyme-linked immunoassay and IL-6 messenger RNA expression levels were determined by real-time polymerase chain reaction. RESULTS: TNF-α induced the phosphorylation of p38 MAP kinase in a time-dependent manner, which was suppressed by the p38 MAP kinase inhibitor, SB203580. TNF-α also stimulated IL-6 release in both a time- and dose-dependent manner. On its own, SB203580 did not stimulate IL-6 secretion from HFCs; however, it dramatically suppressed the degree of IL-6 release stimulated by TNF-α from HFCs. CONCLUSIONS: This is the first report suggesting that TNF-α stimulates the gene expression and protein secretion of IL-6 via p38 MAP kinase in HFCs. A noted association between tissue hypertrophy and inflammation suggests that the p38 MAP kinase inflammatory pathway may be a therapeutic molecular target for LSS.

Polymer fraction including exosomes derived from Chinese hamster ovary cells promoted their growth during serum-free repeated batch culture.

While continuous (perfusion) culture of mammalian cells might reduce the reactor size owing to the high cell density, there is the problem of higher medium cost; however, this problem is expected to be solved by the reuse of growth-promoting components in the culture supernatant. The polymer fraction (PF, 10 kDa-220 nm) collected from the supernatant of serum-free repeated-batch culture of Chinese hamster ovary (CHO) cells in not only adhesion but also suspension promoted the cell growth in respective serum-free cultures. PF contained CD81-positive exosomes and proteins, both of which were necessary for its growth-promoting activity. Consequently, the medium cost for the continuous (perfusion) serum-free suspension culture of CHO cells may be decreased by the repeated collection and addition of PF that contains exosomes and growth factor proteins.

Establishment of 2.5D organoid culture model using 3D bladder cancer organoid culture.

Three-dimensional (3D) organoid culture holds great promises in cancer precision medicine. However, Matrigel and stem cell-stimulating supplements are necessary for culturing 3D organoid cells. It costs a lot of money and consumes more time and effort compared with 2D cultured cells. Therefore, the establishment of cheaper and Matrigel-free organoid culture that can maintain the characteristics of a part of 3D organoids is demanded. In the previous study, we established a dog bladder cancer (BC) 3D organoid culture system by using their urine samples. Here, we successfully isolated cells named "2.5D organoid" from multiple strains of dog BC 3D organoids using 2.5 organoid media. The cell proliferation speed of 2.5D organoids was faster than parental 3D organoid cells. The expression pattern of stem cell markers was close to 3D organoids. Injection of 2.5D organoid cells into immunodeficient mice formed tumors and showed the histopathological characteristics of urothelial carcinoma similar to the injection of dog BC 3D organoids. The 2.5D organoids had a similar sensitivity profile for anti-cancer drug treatment to their parental 3D organoids. These data suggest that our established 2.5D organoid culture method might become a reasonable and useful tool instead of 3D organoids in dog BC research and therapy.

Efficacy of primary liver organoid culture from different stages of non-alcoholic steatohepatitis (NASH) mouse model.

Non-alcoholic steatohepatitis (NASH) is associated with liver fibrosis and cirrhosis, which eventually leads to hepatocellular carcinoma. Although several animal models were developed to understand the mechanisms of NASH pathogenesis and progression, it remains obscure. A 3D organoid culture system can recapitulate organ structures and maintain gene expression profiles of original tissues. We therefore tried to generate liver organoids from different degrees [defined as mild (NASH A), moderate (NASH B) and severe (NASH C)] of methionine- and choline-deficient diet-induced NASH model mice and analyzed the difference of their architecture, cell components, organoid-forming efficacy, and gene expression profiles. Organoids from each stage of NASH model mice were successfully generated. Interestingly, epithelial-mesenchymal transition was observed in NASH C organoids. Expression of Collagen I and an activated hepatic stellite cell marker, α-sma was upregulated in the liver organoids from NASH B and C mice. The analysis of RNA sequencing revealed that several novel genes were upregulated in all NASH liver organoids. These results suggest that our generated liver organoids from different stages of NASH diseased mice might become a useful tool for in vitro studies of the molecular mechanism of NASH development and also for identifying novel biomarkers for early diagnosis of NASH disease.

Characterization of the human E2F4 promoter region and its response to 12-O-tetradecanoylphorbol-13-acetate.

The E2F transcription factors (TFs), which control the progression of the cell cycle in response to DNA-damage and various stresses, are known to interact with a tumour suppressor, Retinoblastoma 1 (RB1). We previously showed that the response of the human RB1 promoter to a 12-O-tetradecanoylphorbol-13-acetate (TPA) in HL-60 cells is mediated by a duplicated GGAA motif, which is also present in the 5'-upstream of the E2F family genes. The motifs are especially rich in the 5'-upstream of the E2F4 gene. In the present study, we constructed luciferase (Luc) expression vectors containing a 466 bp of the 5'-upstream of the human E2F4 gene. The transfection of this plasmid and deletion/mutation-introduced derivatives into HL-60 cells and a Luc reporter assay showed that duplicated and triplicated GGAA (TTCC) motifs in the E2F4 promoter respond to TPA. As expected, electrophoretic mobility shift assay indicated that SPI1 (PU.1) binds to the GGAA motif-containing element. A quantitative RT-PCR and western blotting showed that the E2F4 transcripts and its encoding proteins accumulate during the differentiation of HL-60 into macrophage-like cells. In contrast, the expression of the E2F1 gene and the protein, which possibly acts as a cell cycle accelerator, was greatly diminished.