[Analysis of long-term outcomes of penetrating keratoplasty for congenital corneal opacity].

Objective: To investigate the long-term outcomes of corneal grafts after penetrating keratoplasty(PK) for congenital corneal opacity(CCO) in children aged 0 to 5 years and the related influencing factors. Methods: It was a retrospective series case study. Data of 39 children (55 eyes) who underwent PK surgery due to CCO in the keratology Department of Beijing Tongren Hospital from April 2014 to April 2018 and were followed up for more than 30 months were collected. Among them, there were 17 males (43.6%) and 22 females (56.4%). The age at operation was (16.2±13.3) months, and the follow-up time was (46.4±13.8) months. Clinical data such as basic information, preoperative diagnosis, operation age, operation method and postoperative complications were recorded. The corneal graft transparency was analyzed according to preoperative diagnosis, corneal neovascularization area, age at surgery, monocular or binocular surgery interval, primary surgery type and further surgery, and postoperative complications were observed. Results: At 12 months, 24 months and the last follow-up after PK, 78.2% (43/55), 70.9% (39/55) and 58.2% (32/55) of the affected eyes had clear corneal grafts, respectively.There was no statistical significance between Peters anomaly and sclerocornea (P>0.05), while the extent of neovascularization in the limbus had a significant effect on corneal graft transparency, and graft opacity was more likely to occur in patients with vessel area exceeding 2 quadrants (P<0.05).The highest corneal graft transparency was found in children aged 1 to 3 years 80.8%(21/26) (P<0.05), followed by children younger than 6 months (7/15).The translucency rate of the corneal graft was higher in patients undergoing unilateral surgery than in those undergoing bilateral surgery (P<0.05).Translucency of corneal graft was higher in children with simple surgery than with combined surgery (P<0.05), however, cataract surgery after PK had no significant effect on corneal graft transparency (P>0.05).The postoperative complications mainly included immune rejection in 19 eyes (34.5%), complicated cataract in 13 eyes (23.6%), glaucoma in 7 eyes (13.2%), persistent corneal epithelial defect in 7 eyes (13.2%). Conclusions: After PK in children with CCO, the transparent rate of corneal grafts decreases gradually with time, but the long-term translucency of corneal grafts can still be obtained. The range of corneal neovascularization, age at the time of surgery, whether the surgery was binocular and whether the surgery was combined had an effect on the transparency of corneal graft.

[Corneal melt after intracorneal ring segment implantations: a case report].

A 50-year-old female patient presented to the Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Institute of Ophthalmology, with complaints of right eye pain, tearing, and difficulty opening the eye for over a month after intrastromal corneal ring segment (ICRS) implantation 18 years prior in both eyes. Slit lamp examination revealed corneal stromal melting around the ICRS at the 3 to 4 o'clock position of the right eye, with fluorescein staining. Optical coherence tomography showed epithelial and superficial stromal layer defects in the area of the lesion. The patient was diagnosed with corneal melting after ICRS implantation in the right eye. Under general anesthesia, the corneal stromal ring was removed, and deep lamellar keratoplasty was performed. The patient had no discomfort and the corneal graft remained transparent after the surgery.

[Clinical analysis of penetrating keratoplasty for infants with congenital corneal opacity].

Objective: To investigate the corneal graft survival and related risk factors of primary penetrating keratoplasty in congenital corneal opacity infants. Methods: It was a retrospective cohort study. Data were collected from forty-two infants (51 eyes) who were aged ≤12 months and diagnosed with congenital corneal opacity in Beijing Tongren Hospital and Beijing Anzhen Hospital from January 1, 2017 to January 31, 2018. The mean age at surgery was (5.7±2.2) months (3-12 months). The mean follow-up duration was (28.6±2.6) months (24-33 months). All the patients underwent penetrating keratoplasty. The status of the corneal grafts and complications were observed and recorded during the regular follow-up. The survival probabilities were estimated by using the Kaplan-Meier and Log-rank test. The graft survival between different influence factors was analyzed by using the χ2 test. Results: The Kaplan-Meier survival rates for penetrating keratoplasty were 84.3% (43/51) at 6 months, 78.4% (40/51) at 12 months and 60.8% (31/51) at the last follow-up. The presence of corneal neovascularization was significantly correlated with graft failure (χ²=5.264, P=0.022). The graft survival differed between eyes receiving combined surgery and mere penetrating keratoplasty and in eyes with varied surgical indications (P=0.039, <0.01). Increased intraocular pressure (7 eyes, 13.7%) and persistent epithelial defects (7 eyes, 13.7%) were the most common postoperative complications, followed by complicated cataract (4 eyes, 7.8%) and posterior capsule opacification (2 eyes, 3.9%). Conclusions: The graft survival rate was satisfactory following pediatric keratoplasty although it had a tendency to decrease with the follow-up time. Corneal neovascularization was a major risk factor of graft failure. Surgical indications and procedures also had a certain effect on the graft survival.

[Preliminary observation on the treatment of primary pterygium with modified conjunctival transplantation and amniotic membrane transplantation combined with use of interferon eye drops].

Objective: To observe the clinical effect of modified conjunctival transplantation and amniotic membrane transplantation combined with use of interferon (IFN) alpha-2b eye drops in the treatment of primary pterygium. Methods: This was a prospective case-control study. Patients with primary pterygium were treated from June 1, 2018 to December 31, 2018 in the Department of Ophthalmology, Beijing Tongren Hospital, and they were divided into two groups (the experimental group and the control group) by the method of randomized block design. Patients in the experimental group received modified conjunctival transplantation and amniotic membrane transplantation combined with use of IFN alpha-2b eye drops, while patients in the control group received pterygium resection combined with conjunctival autograft transplantation. The pterygium type and size were observed before operation, while visual acuity, intraocular pressure and anterior segment details were recorded either. The follow-up was done at 1 week, 2 weeks, 1 month, 3 months, 6 months and 12 months after operation. The visual acuity, corneal epithelial defect, and pterygium recurrence were observed. All data in this manuscript are enumeration data, the expected frequency of pterygium type distribution in the two groups was more than 5, and the chi square test was used, fisher's exact test was used to compare the other data between the two groups. Results: Seventy patients (77 eyes) with pterygium were in this study, including 30 males and 40 females, aged from 50-70 years old. There were 35 cases (38 eyes) in the experimental group and 35 cases (39 eyes) in the control group. 12 months after operation there were 54 cases (60 eyes) including 28 cases (30 eyes) in the experimental group and 26 cases (30 eyes) in the control group with complete data. The corneal epithelium defects of 1 eye in each group was repaired within 7-14 days after operation, and the rest eyes were completely repaired within 7 days after operation. There was no significant difference in the distribution of corneal epithelial healing between the two groups (P= 1.00). There was no significant difference between the two groups in the number of eyes distribute with decreased visual acuity (2 eyes in each group), stable visual acuity (15 eyes in the experimental group and 23 eyes in the control group), and improved visual acuity (13 eyes in the experimental group and 5 eyes in the control group) (P=0.053). There was no recurrence in the two groups at 12 months after surgery, and there was no significant difference between the two groups in the number of patients with conjunctival hyperplasia of grades 1, 2 and 3 (P=0.405). Conclusions: Modified conjunctival transplantation and amniotic membrane transplantation combined with use of IFN alpha-2b eye drops got low recurrence rate for primary pterygium and less damage to the healthy conjunctival tissue. This combined treatment strategy provides a new choice for the treatment of pterygium. (Chin J Ophthalmol, 2020, 56: 768-773).

[Clinical observation of autologous simple limbal epithelial transplantation for unilateral limbal stem cell deficiency].

Objective: To discuss effect of autologous simple limbal epithelial transplantation (SLET) performed for unilateral limbal stem cell deficiency (LSCD). Methods: Retrospective case study. In this retrospective study, records of 7 patients (7 eyes) who had undergone autologous SLET for unilateral LSCD, with a minimum of 6 months of follow-up, were reviewed. Demographic details, etiology of LSCD, duration between ocular burn and SLET, prior surgery performed, presence or absence of symblepharon, pre-and post-operative visual acuity, and complications were noted. Results: Seven eyes of 7 patients underwent autologous SLET. With a follow-up of 6 months, a completely epithelialised and stable corneal surface was obtained in all recipient eyes. Visual acuity improved in all patients, while none of the eyes developed any complications. Conclusions: Autologous SLET is an effective and safe modality for treatment of unilateral LSCD. Clinical success rates and visual acuity improvement are equal to or better than those reported with earlier techniques. (Chin J Ophthalmol, 2019, 55:923-927).

[The effectiveness and safety of topical 0.1% tacrolimus after high-risk penetrating keratoplasty].

Objective: To observe the effectiveness and safety of topical 0.1% tacrolimus(FK506) as immunosuppressant in high-risk penetrating corneal transplantation to prevent the immune rejection and to compare the outcomes with topical 1% Cyclosporin A (CsA). Methods: The study consists of 49 patients (50 eyes), who were fitted with the high-risk corneal transplantation standard and undergone the penetrating keratoplasty(PKP) or combined operation in Beijing Tongren hospital between March 2015 to September. With the time sequence, the patients were divided into observation group (FK506 group) and the control group (CsA group). The observation group included 9 females and 16 males with an average age of 57.8±14.8. Twenty-four patients were in the control group (25 eyes), including 10 females and 14 males, with an average age of (45.1±16.2). Observation group was treated with topical 0.1% tacrolimus, and the control group treated with topical 1%CsA. Both groups' treatment combined glucocorticoid as well. Two groups had 1 year follow-up observation. The incidence of rejection was compared by statistical methods of Cox regression. The adverse reactions were graded and compared using Mann-Whitney U test. Results: After one year, 22 cases of the observation group and 23 cases of the control group were accomplished all observations. The rejection rate was 4.54% in observation group and 27.23% in control group. The difference between the groups was statistically significant (χ(2)=4.291, P=0.038). Control group had high rejection rate. Besides, there was no severe side effects happened in both groups. After 1 month after surgery, 36.4% of the observation group showed mild corneal edema. The ratio of mild to moderate corneal edema in the control group was 26.1% and 8.7%. Three months after surgery, 4.5% of the observation group showed mild corneal edema, while 13.0% and 13.0% of the control group was found mild to moderate corneal edema. Six months after surgery, 4.5% of the observation group showed moderate corneal edema. The ratio of mild, moderate to severe corneal edema in the control group was 17.4%, 17.4% and 8.7%. The degree of corneal edema in the control group was more serious in three monthes(Z=-2.770, -2.018, -2.941, P<0.05). The differences in both monthes were statistically significant. Mild neovascularization occurred in the 13.6% of observation group. Mild to severe neovascularization occurred in the 13.0%, 4.3%, and 4.3% control groups. The degree of neovascularization in the control group was higher than that in the observation group(Z=-3.221, P=0.001). The differences in both months were statistically significant. Mild to moderate neovascularization occurred in the 18.2% and 9.1% of observation group. Mild to extremely severe neovascularization occurred in the 17.4%, 26.1%, 4.3% and 4.3% control groups. The degree of neovascularization in the control group was higher than that in the observation group(Z=-1.988, P=0.047).The differences in both monthes were statistically significant. Conclusions: Both 0.1% tacrolimus and 1% cyclosporine A are safe and effective in reducing the rejection after high-risk corneal transplantation. (Chin J Ophthalmol, 2019, 55: 419-427).

[Pay attention to the donor material supply for corneal transplantation].

Corneal transplantation is an important method in the treatment of corneal blindness. It is imperative to improve the treatment effectiveness of corneal disease and reduce the possibility of corneal blindness with the progress of corneal transplantation surgery, the construction and development of eye banks and the rational use of donor materials. This article reviews the component corneal transplantation technology promotion, eye bank construction and preparation of donor slices for component corneal transplantation surgery. (Chin J Ophthalmol, 2016, 52: 641-643).

Tumor suppressive miR-509-5p contributes to cell migration, proliferation and antiapoptosis in renal cell carcinoma.

PURPOSE: The aim of this study was to determine the expression and function of miR-509-5p in renal cell carcinoma (RCC). MATERIALS AND METHODS: In this research, we have conducted quantitative real-time polymerase chain reaction (qRT-PCR) assay to determine the expression level of miR-509-5p in tissues and plasma from renal cell carcinoma patients. We preformed in vitro migration scratch assay, flow cytometry analysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine the exact function of miR-509-5p. RESULTS: We evaluated the expression level of miR-509-5p in RCC tissues and paired adjacent normal tissues from 42 patients and found that miR-509-5p expression in 42 RCC specimens was significantly down-regulated compared to that in adjacent normal tissue. Furthermore, the level of miR-509-5p in RCC patients' plasma was significantly lower than that in control plasma. In addition, the overexpression of miR-509-5p suppressed the proliferation of RCC cell (786-0), induced cell apoptosis and inhibited cell migration in vitro. CONCLUSION: In this study, we have shown that miR-509-5p played an important role in RCC by inhibiting cell proliferation and migration and by promoting cell apoptosis. In addition, miR-509-5p expression was significantly lower in RCC patient plasma compared to that in normal individuals.

Cullin1 is a novel marker of poor prognosis and a potential therapeutic target in human breast cancer.

BACKGROUND: To investigate the role of Cullin1 (Cul1) in the development of breast cancer, we examined the expression of Cul1 in breast cancer tissues and analyzed the correlation between Cul1 expression and clinicopathologic variables and patients survival. PATIENTS AND METHODS: We evaluated the Cul1 expression by immunohistochemistry using a tissue microarray (TMA) which includes 393 breast cancer tissues. We also studied the role of Cul1 in breast cancer cell proliferation, migration and invasion by carrying out CCK8 cell proliferation assay, cell migration and invasion assay. RESULTS: The Cul1 expression was significantly correlated with breast cancer histology grade (P = 0.000), estrogen receptor status (P = 0.001), progesterone receptor status (P = 0.001) and human epidermal growth factor receptor 2 status (P = 0.002). Furthermore, we showed a strong correlation between high Cul1 expression and worse 5-year overall and disease-specific survival rates in breast cancer patients (P = 0.026 and P = 0.015, respectively). Finally, we found that Cul1 knockdown inhibits cell proliferation, migration and invasion abilities. CONCLUSIONS: Cul1 overexpression is significantly correlated with breast cancer progression and predicts worse survival. Cul1 regulates breast cancer cell proliferation, migration and invasion.

Delineation of the cardiac substructures based on PET-CT and contrast-enhanced CT in patients with left breast cancer treated with postoperative radiotherapy.

The aim of this study is to evaluate the volume differences between contrast-enhanced CT-based left ventricle (LV) and PET-CT-based LV and assess the impact of dose on the substructure volume differences in patients with left breast cancer treated with adjuvant radiotherapy. From October 2008 to February 2009, 14 patients with post-operatively confirmed left breast cancer were enrolled in the current study. The patients were scanned using contrast-enhanced CT for simulation, and (18)F-FDG PET-CT was employed to display the structure of the left ventricle of each before radiotherapy (RT). The LV was delineated based on both contrast-enhanced CT and PET-CT. And other substructures, such as the left anterior descending coronary artery (LAD), were contoured in each patient, with the six-field simple intensity modulated radiotherapy (sIMRT) technique created for all. The mean volumes of the left ventricle based on contrast-enhanced CT (LV-CT) and PET-CT (LV-PET) were found to be 107.296 cm(3) and 112.931 cm(3), respectively (p = 0.06). The volume of LV receiving ≥ 50% prescription dose was significantly correlated with the volume of the heart receiving the same dosage (γ = 0.869). There was less correlation between the volume of LAD and that of the heart under the same condition (γ = 0.22). As a conclusion, the left ventricle can be delineated effectively based on the image of PET-CT, the contrast-enhanced CT based LV can serve as an appropriate alternative. Moreover, the volume of LV receiving high dose in RT closely correlated with the volume of the heart using sIMRT technique, which may pave the way for further exploring radiation-induced cardiac injuries in patients with left breast cancer.

The effects of glaucoma filtering surgery on anterior chamber-associated immune deviation and contribution of lymphatic drainage in rats.

PURPOSE: To investigate whether glaucoma filtering surgery (GFS) in rats would impair the eye's capacity to induce anterior chamber-associated immune deviation (ACAID) and assess the possible mechanism involved. METHODS: Rats subjected to GFS were injected with bovine serum antigen (BSA) into the anterior chamber to induce ACAID. Animals that had their cervical lymph nodes (CLNs) excised before filtering surgery and those that had sham filtering surgery served as control comparison groups. Antigen-specific delayed-type hypersensitivity (DTH) was used to identify the induction of ACAID. Antigen level in the CLNs was indicated by the percentage of FITC-positive cells in CLNs after FITC dextran was injected into the anterior chamber. Statistical analyses were performed using the student's t-test for comparison of data between control and experimental groups. A P<0.05 was required for results to be considered statistically significant. RESULTS: Rats undergoing GFS demonstrated antigen-specific DTH, while those in the sham filtering surgery or CLNs excised groups failed to acquire an antigen-specific DTH response. The percentage of FITC-positive cells in CLNs was significantly increased (P=0.001) in GFS (mean+/-SD: 2.96+/-0.67%) vs controls (1.57+/-0.48%) at 1 day, but not at 3, 5, 7, or 12 days post-antigen injection. CONCLUSIONS: GFS prevents the induction of ACAID in rats, and the antigen drainage to CLNs plays a critical role in this process. The results suggest that the ocular immune status might be altered by GFS.

The RING domains of the promyelocytic leukemia protein PML and the arenaviral protein Z repress translation by directly inhibiting translation initiation factor eIF4E.

The promyelocytic leukemia protein (PML) is a mammalian regulator of cell growth which is characteristically disrupted in acute promyelocytic leukemia and by a variety of viruses. PML contains a RING domain which is required for its growth-suppressive and antiviral properties. Although normally nuclear, in certain pathogenic conditions, including arenaviral infection, PML is relocated to the cytoplasm, where its functions are poorly understood. Here, we observe that PML and arenavirus protein Z use regions around the first zinc-binding site of their respective RING domains to directly interact, with sub-micromolar affinity, with the dorsal surface of translation initiation factor eIF4E, representing a novel mode of eIF4E recognition. PML and Z profoundly reduce the affinity of eIF4E for its substrate, the 5' 7-methyl guanosine cap of mRNA, by over 100-fold. Association with the dorsal surface of eIF4E and direct antagonism of mRNA cap binding by PML and Z lead to direct inhibition of translation. These activities of the RING domains of PML and Z do not involve ubiquitin-mediated protein degradation, in contrast to many RINGs which have been observed to do so. Although PML and Z have well characterized physiological functions in regulation of growth and apoptosis, this work establishes the first discrete biochemical mechanism which underlies the biological activities of their RING domains. Thus, we establish PML and Z as translational repressors, with potential contributions to the pathogenesis of acute promyelocytic leukemia and variety of viral infections.

SUMO-1 modification of Mdm2 prevents its self-ubiquitination and increases Mdm2 ability to ubiquitinate p53.

Mdm2 is an E3 ubiquitin ligase for the p53 tumor suppressor protein. We demonstrate that Mdm2 is conjugated with SUMO-1 (sumoylated) at Lys-446, which is located within the RING finger domain and plays a critical role in Mdm2 self-ubiquitination. Whereas mutant Mdm2(K446R) is stabilized, it elicits increased degradation of p53 and concomitant inhibition of p53-mediated apoptosis. In vitro sumoylation of Mdm2 abrogates its self-ubiquitination and increases its ubiquitin ligase activity toward p53. Radiation caused a dose- and time-dependent decrease in the degree of Mdm2 SUMO-1 modification, which is inversely correlated with the levels of p53. Our results suggest that the maintenance of the intrinsic activity of a RING finger E3 ubiquitin ligase is sumoylation dependent and that reduced Mdm2 sumoylation in response to DNA damage contributes to p53 stability.

The conserved RING-H2 finger of ROC1 is required for ubiquitin ligation.

ROC1 is a common component of a large family of ubiquitin E3 ligases that regulate cell cycle progression and signal transduction pathways. Here we present evidence suggesting that a conserved RING-H2 structure within ROC1 is critical for its ubiquitin ligation function. Mercury-containing sulfhydryl modification agents (rho-hydroxymercuribenzoate and mercuric chloride) irreversibly inhibit the ROC1-CUL1 ubiquitin ligase activity without disrupting the complex. Consistent with this, these reagents also eliminate the ability of the Skp1-CUL1-HOS-ROC1 E3 ligase complex to support the ubiquitination of IkappaBalpha. Site-directed mutagenesis analysis identifies RING-H2 finger residues Cys(42), Cys(45), Cys(75), His(77), His(80), Cys(83), Cys(94), and Asp(97) as being essential for the ROC1-dependent ubiquitin ligase activity. Furthermore, C42S/C45S and H80A mutations reduce the ability of ROC1 to interact with CUL1 in transfected cells and diminish the capacity of ROC1-CUL1 to form a stable complex with Cdc34 in vitro. However, C75S, H77A, C94S, and D97A substitutions have no detectable effect on ROC1 binding activities. Thus, the ROC1 RING-H2 finger may possess multiple biochemical properties that include stabilizing an interaction with CUL1 and recruiting Cdc34. A possible role of the RING finger in facilitating the Ub transfer reaction is discussed.

The SCF(HOS/beta-TRCP)-ROC1 E3 ubiquitin ligase utilizes two distinct domains within CUL1 for substrate targeting and ubiquitin ligation.

We describe a purified ubiquitination system capable of rapidly catalyzing the covalent linkage of polyubiquitin chains onto a model substrate, phosphorylated IkappaBalpha. The initial ubiquitin transfer and subsequent polymerization steps of this reaction require the coordinated action of Cdc34 and the SCF(HOS/beta-TRCP)-ROC1 E3 ligase complex, comprised of four subunits (Skp1, cullin 1 [CUL1], HOS/beta-TRCP, and ROC1). Deletion analysis reveals that the N terminus of CUL1 is both necessary and sufficient for binding Skp1 but is devoid of ROC1-binding activity and, hence, is inactive in catalyzing ubiquitin ligation. Consistent with this, introduction of the N-terminal CUL1 polypeptide into cells blocks the tumor necrosis factor alpha-induced and SCF-mediated degradation of IkappaB by forming catalytically inactive complexes lacking ROC1. In contrast, the C terminus of CUL1 alone interacts with ROC1 through a region containing the cullin consensus domain, to form a complex fully active in supporting ubiquitin polymerization. These results suggest the mode of action of SCF-ROC1, where CUL1 serves as a dual-function molecule that recruits an F-box protein for substrate targeting through Skp1 at its N terminus, while the C terminus of CUL1 binds ROC1 to assemble a core ubiquitin ligase.

HOS, a human homolog of Slimb, forms an SCF complex with Skp1 and Cullin1 and targets the phosphorylation-dependent degradation of IkappaB and beta-catenin.

SCF E3 ubiquitin ligases mediate ubiquitination and proteasome-dependent degradation of phosphorylated substrates. We identified a human F-box/WD40 repeats protein (HOS), which is homologous to Slimb/h betaTrCP. Being a part of SCF complex with Skp1 and Cullin1, HOS specifically interacted with the phosphorylated IkappaB and beta-catenin, targeting these proteins for proteasome-dependent degradation in vivo. This targeting required Cullin1 as expression of a mutant Cullin1 abrogated the degradation of IkappaB and of beta-catenin. Mutant HOS which lacks the F-box blocked TNF alpha-induced degradation of IkappaB as well as GSK3beta-mediated degradation of beta-catenin. This mutant also inhibited NF-kappaB transactivation and increased the beta-catenin-dependent transcription activity of Tcf. These results demonstrate that SCF(HOS) E3 ubiquitin ligase regulate both NF-kappaB and beta-catenin signaling pathways.

Suppression of cell transformation by the cyclin-dependent kinase inhibitor p57KIP2 requires binding to proliferating cell nuclear antigen.

Proper control of the mammalian cell cycle requires the function of cyclin-dependent kinase (CDK) inhibitors. The p21 family currently includes three distinct genes, p21, p27(Kip1), and p57(Kip2), that share a common N-terminal domain for binding to and inhibiting the kinase activity of CDK-cyclin complexes. The p21 protein also binds to proliferating cell nuclear antigen (PCNA) through a separate C-terminal domain affecting DNA replication and repair. The p27 and p57 proteins also each contain unique C-terminal domains whose functions are unknown. Here we show that the human p57 protein, like p21, contains a PCNA-binding domain within its C terminus that, when separated from its N-terminal CDK-cyclin binding domain, can prevent DNA replication in vitro and S phase entry in vivo. Disruption of either CDK/cyclin or PCNA binding partially reduced p57's ability to suppress myc/RAS-mediated transformation in primary cells, while loss of both inhibitory functions completely eliminated p57's suppressive activity. Thus, control of cell cycle and suppression of cell transformation by p57 require both CDK and PCNA inhibitory activity, and disruption of either or both functions may lead to uncontrolled cell growth.

p53 is phosphorylated by CDK7-cyclin H in a p36MAT1-dependent manner.

The tumor suppressor protein p53 acts as a transcriptional activator that can mediate cellular responses to DNA damage by inducing apoptosis and cell cycle arrest. p53 is a nuclear phosphoprotein, and phosphorylation has been proposed to be a means by which the activity of p53 is regulated. The cyclin-dependent kinase (CDK)-activating kinase (CAK) was originally identified as a cellular kinase required for the activation of a CDK-cyclin complex, and CAK is comprised of three subunits: CDK7, cyclin H, and p36MAT1. CAK is part of the transcription factor IIH multiprotein complex, which is required for RNA polymerase II transcription and nucleotide excision repair. Because of the similarities between p53 and CAK in their involvement in the cell cycle, transcription, and repair, we investigated whether p53 could act as a substrate for phosphorylation by CAK. While CDK7-cyclin H is sufficient for phosphorylation of CDK2, we show that p36MAT1 is required for efficient phosphorylation of p53 by CDK7-cyclin H, suggesting that p36MAT1 can act as a substrate specificity-determining factor for CDK7-cyclin H. We have mapped a major site of phosphorylation by CAK to Ser-33 of p53 and have demonstrated as well that p53 is phosphorylated at this site in vivo. Both wild-type and tumor-derived mutant p53 proteins are efficiently phosphorylated by CAK. Furthermore, we show that p36 and p53 can interact both in vitro and in vivo. These studies reveal a potential mechanism for coupling the regulation of p53 with DNA repair and the basal transcriptional machinery.

Transcription-positive cofactor 4 forms complexes with HSSB (RPA) on single-stranded DNA and influences HSSB-dependent enzymatic synthesis of simian virus 40 DNA.

The replication of simian virus 40 (SV40) DNA in vitro requires a trimeric single-stranded DNA (ssDNA)-binding protein called HSSB or RPA. HSSB supports the unwinding of DNA containing the SV40 origin in the presence of the viral-encoded T antigen and is required for the initiation of RNA primer synthesis as well as processive elongation of DNA catalyzed by the DNA polymerase delta holoenzyme. In this report we show that the transcription positive cofactor 4 (PC4), a ssDNA-binding protein, forms complexes with HSSB on ssDNA and markedly affects the replication functions of HSSB. PC4 supports T antigen-catalyzed unwinding of SV40 origins in lieu of HSSB but inhibits both RNA primer synthesis and polymerase delta-catalyzed DNA chain elongation reactions. These inhibitory effects can be reversed by the addition of excess HSSB. Depending on the concentration of HSSB, PC4 is capable of either inhibiting or activating SV40 DNA replication measured in both mono- and dipolymerase systems. The possible role of PC4 in the initiation of DNA replication is discussed.

Studies on the in vitro phosphorylation of HSSB-p34 and -p107 by cyclin-dependent kinases. Cyclin-substrate interactions dictate the efficiency of phosphorylation.

Cyclin-dependent kinases (Cdks) are required for cell cycle progression. Two potentially significant Cdk substrates in human cells are the human single-stranded binding protein (HSSB or RPA), which plays an essential role in DNA replication, repair, and recombination, and the tumor suppressor p107 which acts to negatively regulate cell growth. In this report we describe the in vitro phosphorylation of these two proteins by Cdks in an attempt to understand how cyclin-substrate interactions direct phosphorylation efficiencies. We show that cyclin A-Cdk2 efficiently phosphorylates the p34 subunit of HSSB (HSSB-p34) alone or as a part of the heterotrimeric complex. In contrast, cyclin E-Cdk2 that is active in phosphorylating histone H1, does not support the phosphorylation of the p34 subunit of HSSB. We provide evidence that this differential phosphorylation results from a specific interaction between HSSB-p34 and cyclin A, but not cyclin E. Thus the observed cell cycle-dependent phosphorylation of HSSB-p34 at the G1 to S transition is most likely catalyzed by cyclin A-Cdk2 initiated by the direct interaction between cyclin A and the HSSB-p34 subunit. These studies are consistent with our previous observation that p107, which directly binds cyclin A, is efficiently phosphorylated by cyclin A-Cdk2 but not cyclin B-associated kinases. Here we further demonstrate that cyclin A only complexes with p107 in its unphosphorylated form. These data suggest a catalytic mechanism by which Cdk acts: substrate targeting by a cyclin-substrate interaction followed by dissociation of the Cdk upon phosphate incorporation allowing the Cdk to become available for the next cycle of phosphorylation.