George Zachos

Expression of ras proto-oncogenes: regulation and implications in the development of human tumors

2. Transcriptional regulation of the human c-H-rasl gene 66 2.1. Regulation of the H-ras gene expression from promoter-like sequences 66 2.2. Regulation of the H-ras gene expression from intronic sequences 67 2.3. The role of the VTR in expression of the H-ras gene 68 2.4. DNA methylation affecting H-ras gene expression. 68 2.5. Expression of the H-ras proto-oncogene is controlled by alternative splicing. 68

Phosphorylation at serine 331 is required for Aurora B activation

Chk1 phosphorylates Aurora B to promote its full activation during mitotic prometaphase.

Chk2 is required for optimal mitotic delay in response to irradiation-induced DNA damage incurred in G2 phase

Whether Chk2 contributes to DNA damage-induced arrest in G2 has been controversial. To investigate this issue further, we generated Chk2-deficient DT40 B-lymphoma cells by gene targeting and compared their cell cycle response to ionizing radiation (IR) with wild-type (WT) and isogenic Chk1-deficient counterparts. After moderate doses of IR (4u2009Gy), we find that Chk2−/− cells which are in G1 or S phase at the time of irradiation arrest efficiently in G2. In contrast, Chk2−/− cells which are in G2 when DNA damage is incurred exhibit an impaired mitotic delay compared to WT, with the result that cells enter mitosis with damaged DNA as judged by the presence of numerous γ-H2AX foci on condensed chromosomes. Impaired G2 delay as the result of Chk2 deficiency can be detected at very low doses of radiation (0.1u2009Gy), and may allow division with spontaneous DNA damage, since a higher proportion of mitotic Chk2−/− cells bear spontaneous γ-H2AX foci and damaged chromosomes during unperturbed growth compared to WT. The contribution of Chk2 to G2/M delay is epistatic to that of Chk1, since Chk1−/− cells exhibit no measurable mitotic delay at any radiation dose tested. We suggest that this function of Chk2 could contribute to tumour suppression, since cell division with low levels of spontaneous damage is likely to promote genetic instability and thus carcinogenesis.

Transcriptional regulation of the c-H-ras1 gene by the P53 protein is implicated in the development of human endometrial and ovarian tumours

The human c-H-ras1 gene contains within the first intron a p53 element, which functions as a transcriptional enhancer. Using nuclear extracts from human endometrial and ovarian tumours in gel retardation assays, we examined the binding levels of the P53 protein to the H-ras element in tumour versus the adjacent normal tissue. Elevated P53 binding in the tumour tissue was found in 5/12 (42%) endometrial and in 2/5 (40%) ovarian specimens and these cases were found to overexpress wild-type P53. Loss of P53 binding to the H-ras element due to p53 mutations, was observed in 3/12 (25%) endometrial and in 1/5 (20%) ovarian cases. Similar P53 binding levels to the H-ras element were found in 4/12 (33%) endometrial and in 2/5 (40%) ovarian pairs showing normal expression of wild-type P53. Overexpression of the Ras p21 protein correlated with elevated binding and increased nuclear levels of wild-type P53. Our results suggest that P53 protein alterations, participate in the development of human gynecological neoplasias through aberrant transcriptional regulation of the H-ras proto-oncogene.

Clks 1, 2 and 4 prevent chromatin breakage by regulating the Aurora B-dependent abscission checkpoint

When chromatin is trapped at the intercellular bridge, cells delay completion of cytokinesis (abscission) to prevent chromosome breakage. Here we show that inhibition of Cdc-like kinases (Clks) 1, 2 or 4 accelerates midbody resolution in normally segregating cells and correlates with premature abscission, chromatin breakage and generation of DNA damage in cytokinesis with trapped chromatin. Clk1, Clk2 and Clk4 localize to the midbody in an interdependent manner, associate with Aurora B kinase and are required for Aurora B–serine 331 (S331) phosphorylation and complete Aurora B activation in late cytokinesis. Phosphorylated Aurora B–S331 localizes to the midbody centre and is required for phosphorylation and optimal localization of the abscission protein Chmp4c. In addition, expression of phosphomimetic mutants Aurora B–S331E or Chmp4c-S210D delays midbody disassembly and prevents chromatin breakage in Clk-deficient cells. We propose that Clks 1, 2 and 4 impose the abscission checkpoint by phosphorylating Aurora B–S331 at the midbody.

Chk1 protects against chromatin bridges by constitutively phosphorylating BLM serine 502 to inhibit BLM degradation

ABSTRACT Chromatin bridges represent incompletely segregated chromosomal DNA connecting the anaphase poles and can result in chromosome breakage. The Blooms syndrome protein helicase (BLM, also known as BLMH) suppresses formation of chromatin bridges. Here, we show that cells deficient in checkpoint kinase 1 (Chk1, also known as CHEK1) exhibit higher frequency of chromatin bridges and reduced BLM protein levels compared to controls. Chk1 inhibition leads to BLM ubiquitylation and proteasomal degradation during interphase. Furthermore, Chk1 constitutively phosphorylates human BLM at serine 502 (S502) and phosphorylated BLM localises to chromatin bridges. Mutation of S502 to a non-phosphorylatable alanine residue (BLM-S502A) reduces the stability of BLM, whereas expression of a phospho-mimicking BLM-S502D, in which S502 is mutated to aspartic acid, stabilises BLM and prevents chromatin bridges in Chk1-deficient cells. In addition, wild-type but not BLM-S502D associates with cullin 3, and cullin 3 depletion rescues BLM accumulation and localisation to chromatin bridges after Chk1 inhibition. We propose that Chk1 phosphorylates BLM-S502 to inhibit cullin-3-mediated BLM degradation during interphase. These results suggest that Chk1 prevents deleterious anaphase bridges by stabilising BLM.

Chk2 prevents mitotic exit when the majority of kinetochores are unattached.

As an essential step of the spindle checkpoint, Chk2 stabilizes Mps1 and phosphorylates Aurora B to prevent mitotic exit when most kinetochores remain unattached.

Glucocorticoid and Estrogen Receptors Have Elevated Activity in Human Endometrial and Ovarian Tumors as Compared to the Adjacent Normal Tissues and Recognize Sequence Elements of the H‐ras Proto‐oncogene

We examined the level of receptor binding in H‐ras elements, using nuclear extracts derived from human endometrial and ovarian lesions and from adjacent normal tissue in gel retardation assays. We found increased binding of the glucocorticoid receptor (GR) to the H‐ras GR element in more than 90% of endometrial tumors and in all ovarian tumors tested, as compared to the corresponding adjacent normal tissue. Additionally, we found elevated binding of the estrogen receptor (ER) in H‐ras ER element in all pairs of ovarian tumor/normal tissue tested, whereas in ER‐negative control breast tumor/normal tissue pairs, no differences in ER DNA‐binding levels were observed. These results suggest that steroid hormone receptor binding could directly activate the H‐ras oncogenic potency in human endometrial and ovarian lesions, providing additional evidence for the role of H‐ras expression in hormonally responsive human cancers.

Chk1 and Mps1 jointly regulate correction of merotelic kinetochore attachments

Summary If uncorrected, merotelic kinetochore attachments can induce mis-segregated chromosomes in anaphase. We show that checkpoint kinase 1 (Chk1) protects vertebrate cells against merotelic attachments and lagging chromosomes and is required for correction of merotelic attachments during a prolonged metaphase. Decreased Chk1 activity leads to hyper-stable kinetochore microtubules, unstable binding of MCAK, Kif2b and Mps1 to centromeres or kinetochores and reduced phosphorylation of Hec1 by Aurora-B. Phosphorylation of Aurora-B at serine 331 (Ser331) by Chk1 is high in prometaphase and decreases significantly in metaphase cells. We propose that Ser331 phosphorylation is required for optimal localization of MCAK, Kif2b and Mps1 to centromeres or kinetochores and for Hec1 phosphorylation. Furthermore, inhibition of Mps1 activity diminishes initial recruitment of MCAK and Kif2b to centromeres or kinetochores, impairs Hec1 phosphorylation and exacerbates merotelic attachments in Chk1-deficient cells. We propose that Chk1 and Mps1 jointly regulate Aurora-B, MCAK, Kif2b and Hec1 to correct merotelic attachments. These results suggest a role for Chk1 and Mps1 in error correction.

The ESCRT protein Chmp4c regulates mitotic spindle checkpoint signaling

The mitotic spindle checkpoint delays anaphase onset in the presence of unattached kinetochores, and efficient checkpoint signaling requires kinetochore localization of the Rod–ZW10–Zwilch (RZZ) complex. In the present study, we show that human Chmp4c, a protein involved in membrane remodeling, localizes to kinetochores in prometaphase but is reduced in chromosomes aligned at the metaphase plate. Chmp4c promotes stable kinetochore–microtubule attachments and is required for proper mitotic progression, faithful chromosome alignment, and segregation. Depletion of Chmp4c diminishes localization of RZZ and Mad1-Mad2 checkpoint proteins to prometaphase kinetochores and impairs mitotic arrest when microtubules are depolymerized by nocodazole. Furthermore, Chmp4c binds to ZW10 through a small C-terminal region, and constitutive Chmp4c kinetochore targeting causes a ZW10-dependent checkpoint metaphase arrest. In addition, Chmp4c spindle functions do not require endosomal sorting complex required for transport–dependent membrane remodeling. These results show that Chmp4c regulates the mitotic spindle checkpoint by promoting localization of the RZZ complex to unattached kinetochores.