Grady F. Saunders

Mir-17-5p Regulates Breast Cancer Cell Proliferation by Inhibiting Translation of AIB1 mRNA

ABSTRACT MicroRNAs are an extensive family of ∼22-nucleotide-long noncoding RNAs expressed in a wide range of eukaryotes, including humans, and they are important in development and disease. We found that microRNA Mir-17-5p has extensive complementarity to the mRNA of AIB1 (named for “amplified in breast cancer 1”). Cell culture experiments showed that AIB1 expression was downregulated by Mir-17-5p, primarily through translational inhibition. Expression of Mir-17-5p was low in breast cancer cell lines. We also found that downregulation of AIB1 by Mir-17-5p resulted in decreased estrogen receptor-mediated, as well as estrogen receptor-independent, gene expression and decreased proliferation of breast cancer cells. Mir-17-5p also completely abrogated the insulin-like growth factor 1-mediated, anchorage-independent growth of breast cancer cells. Our results reveal that Mir-17-5p has a role as a tumor suppressor in breast cancer cells.

LOCALIZATION OF SINGLE COPY DNA SEQUENCES ON G-BANDED HUMAN CHROMOSOMES BY IN SITU HYBRIDIZATION

Recombinant lambda bacteriophage clone H3 containing a human DNA segment of 14.9 kb present in one or two copies per haploid genome was isolated. In situ hybridization to human metaphase chromosomes of the 3H-labeled cloned DNA resulted in highly significant labeling (53% of cells) of band p36 of chromosome 1, such that 22% of all chromosomal grains were located on this region. Hybridization was dependent upon the presence of dextran sulfate in the hybridization mixture and was not affected by repetitive DNA competitor. These results demonstrate localization of a single copy sequence on human metaphase chromosomes.

Molecular dissection of Pax6 function: the specific roles of the paired domain and homeodomain in brain development

The transcription factor Pax6 plays a key role during development of various organs, including the brain where it affects cell fate, cell proliferation and patterning. To understand how Pax6 coordinates these diverse effects at the molecular level, we examined the role of distinct DNA-binding domains of Pax6, the homeodomain (HD), the paired domain (PD) and its splice variant (5a), using loss- and gain-of-function approaches. Here we show that the PD is necessary for the regulation of neurogenesis, cell proliferation and patterning effects of Pax6, since these aspects are severely affected in the developing forebrain of the Pax6Aey18 mice with a deletion in the PD but intact homeo- and transactivation domains. In contrast, a mutation of the HD lacking DNA-binding (Pax64Neu) resulted in only subtle defects of forebrain development. We further demonstrate distinct roles of the two splice variants of the PD. Retrovirally mediated overexpression of Pax6 containing exon 5a inhibited cell proliferation without affecting cell fate, while Pax6 containing the canonical form of the PD lacking exon 5a affected simultaneously cell fate and proliferation. These results therefore demonstrate a key role of the PD in brain development and implicate splicing as a pivotal factor regulating the potent neurogenic role of Pax6.

Androgen receptor polymorphisms: Association with prostate cancer risk, relapse and overall survival

Several reports have suggested that one or both of the trinucleotide repeat polymorphisms in the human androgen receptor (hAR) gene, (CAG)n coding for polyglutamine and (GGC)ncoding for polyglycine, may be associated with prostate cancer risk; but no study has investigated their association with disease progression. We present here a study of both hAR trinucleotide repeat polymorphisms not only as they relate to the initial diagnosis but also as they are associated with disease progression after therapy. Lymphocyte DNA samples from 178 British Caucasian prostate cancer patients and 195 control individuals were genotyped by PCR for the (CAG)n and (GGC)n polymorphisms in hAR. Univariate Cox proportional hazard analysis indicated that stage, grade and GGC repeat length were individually significant factors associated with disease‐free survival (DFS) and overall survival (OS). The relative risk (RR) of relapse for men with more than 16 GGC repeats was 1.74 (95% CI 1.08–2.79) and of dying from any cause, 1.98 (1.13–3.45). Adjusting for stage and grade, GGC effects remained but were not significant (RRDFS= 1.60, p = 0.052; RROS= 1.65, p = 0.088). The greatest effects were in stage T1‐T2 (RRDFS= 3.56, 95% CI 1.13–11.21) and grade 1 (RRDFS= 6.47, 95% CI 0.57–72.8) tumours. No differences between patient and control allele distributions were found by odds‐ratio analysis, nor were trends with stage or grade evident in the proportion of short CAG alleles. Non‐significant trends with stage and grade were found in the proportion of short GGC alleles. The (GGC)n polymorphism in this population is a significant predictor of disease outcome. Since the (GGC)n effect is strongest in early‐stage tumours, this marker may help forecast aggressive behaviour and could be used to identify those patients meriting more radical treatment. Int. J. Cancer (Pred. Oncol.) 84:458–465, 1999.

Truncation Mutations in the Transactivation Region of PAX6 Result in Dominant-Negative Mutants

PAX6 is a transcription factor with two DNA-binding domains (paired box and homeobox) and a proline-serine-threonine (PST)-rich transactivation domain. PAX6 regulates eye development in animals ranging from jellyfish toDrosophila to humans. Heterozygous mutations in the humanPAX6 gene result in various phenotypes, including aniridia, Peter’s anomaly, autosomal dominant keratitis, and familial foveal dysplasia. It is believed that the mutated allele ofPAX6 produces an inactive protein and aniridia is caused due to genetic haploinsufficiency. However, several truncation mutations have been found to occur in the C-terminal half ofPAX6 in patients with Aniridia resulting in mutant proteins that retain the DNA-binding domains but have lost most of the transactivation domain. It is not clear whether such mutants really behave as loss-of-function mutants as predicted by haploinsufficiency. Contrary to this theory, our data showed that these mutants are dominant-negative in transient transfection assays when they are coexpressed with wild-type PAX6. We found that the dominant-negative effects result from the enhanced DNA binding ability of these mutants. Kinetic studies of binding and dissociation revealed that various truncation mutants have 3–5-fold higher affinity to various DNA-binding sites when compared with the wild-type PAX6. These results provide a new insight into the role of mutant PAX6 in causing aniridia.

Iris hypoplasia in mice that lack the alternatively spliced Pax6(5a) isoform

PAX6 is an evolutionarily conserved transcription factor that plays a critical role in vertebrate and invertebrate eye formation. Heterozygous null mutations in the PAX6 gene result in aniridia in humans and a distinct small eye syndrome in rodents. Vertebrates primarily express two alternatively spliced isoforms of Pax6 that differ by the presence or absence of exon 5a (e5A) that encodes an additional 14 aa residues within the paired domain. The e5a-containing isoform, PAX6(5a), is specific to and conserved in vertebrates. To determine the role of PAX6(5a), we have generated mice that lack e5a of the Pax6 gene. Unlike Pax6 null mice that exhibit anopthalmia with central nervous system defects and lethality, 5a isoform-null mice have iris hypoplasia and defects in the cornea, lens, and retina. Although invertebrates have structures that respond to light intensity and act to restrict light exposure of the eyes, a significant and distinct feature of the vertebrate eye is its ability to regulate the amount of incoming light through contractile pupils. This feature of the eye not only allows vertebrates to see in various light conditions but also enhances image resolution. The requirement of the 5a isoform in iris formation suggests that the evolution of this isoform contributed to advanced features of the vertebrate eye.

Localization of sequences specifying messenger RNA to light-staining G-bands of human chromosomes

Total cytoplasmic polyadenylated RNA was isolated from the human lymphocyte cell line Wil2 by oligo(dT)-cellulose chromatography. Tritiated complementary DNA (cDNA) was transcribed from the RNA and used as a probe for in situ hybridization to metaphase chromosomes. The majority of G-negative or lightly staining bands were found to be preferential sites of hybridization.

PAX6, Paired Domain Influences Sequence Recognition by the Homeodomain

PAX6 functions as a transcription factor and has two DNA-binding domains, a paired domain (PD) and a homeodomain (HD), joined by a glycine-rich linker and followed by a proline-serine-threonine-rich (PST) transactivation region at the C terminus. The mechanism of PAX6 function is not clearly understood, and few target genes in vertebrates have been identified. In this report we described the functional analyses of patient missense mutations from the paired domain region of PAX6 and a paireddomain-less isoform (PD-less) of Pax6 that lacks the paired domain and part of the glycine-rich linker. The PD-less was expressed in the brain, eyes, and pancreas of mouse. The level of expression of this isoform was relatively higher in brain. The mutation sites PAX6-L46R and -C52R were located in the PD of PAX6 on either end of the 5a-polypeptide insert of the alternatively spliced form of PAX6, PAX6-5a. Another PAX6 mutant V53L described in this report was adjacent to C52R. We created corresponding mutations in PAX6 and PAX6-5a, and evaluated their transcriptional activation and DNA binding properties. The PD mutants of PAX6 (L46R, C52R, and V53L) exhibited lower transactivation activities and variable DNA binding ability than wild-type PAX6 with PD DNA-binding consensus sequences. The mutated amino acids containing PAX6-5a isoforms showed unexpected transactivation properties with a reporter containing HD DNA-binding sequences. PAX6-5a-C52R, and -V53L showed lower transactivation activities, but PAX6-5a-L46R had greater transactivation ability than PAX6-5a. The PD-less isoform of Pax6 lost its transactivational ability but could bind to the HD DNA-binding sequences. Functional analysis of the PD-less isoform of Pax6 as well as findings related to missense mutations in the PD suggest that the PD of PAX6 is required for HD function.

Small eye (Sey): Cloning and characterization of the murine homolog of the human aniridia gene

Phenotypic parallels and genetic evidence from comparative mapping suggest that the murine Small eye (Sey) and human aniridia (AN) disorders are homologous. This report describes the isolation of a murine embryonic cDNA that is structurally homologous to the AN cDNA were recently cloned. The murine cDNA detects a 2.7-kb transcript in the adult mouse eye and cerebellum and in human glioblastomas, suggesting a neuroectodermal involvement in the etiology of Sey/AN. Sequence comparison between the murine and the human cDNAs revealed extensive homology in nucleotide sequence (greater than 92%) and virtual identity at the amino acid level. None of the differing amino acids was located within the paired box and homeobox DNA-binding domains. These results provide evidence for a common molecular basis underlying the two genetic disorders and suggest that the Sey system would be an authentic model for human AN.

WT1 and GATA1 expression in myelodysplastic syndrome and acute leukemia

The Wilms’ tumor protein, WT1, represses transcription from several growth factor genes. WT1 transcription is regulated in erythroid and myeloid lineages by the transcription factor GATA-1. Using a sensitive, isotopic duplex RT-PCR procedure amplifying WT1 or GATA-1 together with β-actin as the internal control in a single reaction mix, we quantitated the expression of WT1 and GATA-1 mRNA of 16 patients with myelodysplastic syndrome (MDS), 56 with acute myeloid leukemia (AML) and 22 with acute lymphoblastic leukemia (ALL). K562 was used as reference positive control for this cell line expresses both WT1 and GATA-1. Among MDS patients, increased WT1 expression was found in refractory anemia with excess blast (RAEB) and RAEB in transformation (RAEB-T) subtypes compared to the normal controls, whereas WT1 expression in refractory anemia (RA) was not different from the normal control level. All of AML cases of subtypes M0, M1, M2 and M3 expressed WT1 more than three times the normal WT1 level. Subtypes M4 to M7 showed significantly lower WT1 levels than M1 to M3 and AML cases with CD14+ expressed less WT1 than CD14−. Higher than normal WT1 levels were also expressed in cases of ALL.