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Brenda L Gallie

Publications and source records attributed to Brenda L Gallie.

At least 19 recordsLinked to original sources
PubMed2007

Human retinoblastoma is not caused by known pRb-inactivating human DNA tumor viruses.

Retinoblastomas occur as the consequence of inactivation of the tumor suppressor retinoblastoma protein (pRb), classically upon biallelic inactivation of the RB1 gene locus. Recently, human papillomavirus (HPV) genomic DNA has been detected in retinoblastomas. To investigate the possibility that oncoproteins encoded by pRb-inactivating DNA tumor viruses play a role in the pathogenesis of human retinoblastoma, 40 fresh-frozen tumors were analyzed for the presence of HPV, adenovirus (HAdV) and polyomavirus (BKV, JCV and SV40) genomic DNA sequences by real-time polymerase chain reaction (PCR). Tumors were screened for genetic and epigenetic alterations in all 27 exons of the RB1 gene locus and promoter by exonic copy number detection, sequencing and methylation-specific PCR of the promoter region. Retinoblastoma tumors from children with bilateral familial (n=1), bilateral nonfamilial (n=1) and unilateral nonfamilial (n=38) disease were analyzed. Inactivating modifications to the RB1 gene locus were identified on both the alleles in 27 tumors, one allele in 8, and neither allele in 5 cases. A median of over 107,000 tumor cells were analyzed for viral genomic DNA in each PCR reaction. All tumor samples were negative for 37 HPV types, 51 HAdV types, BKV and JCV genomic sequences. Very low copy number (0.2-260 copies per 100,000 tumor cells) SV40 genomic DNA detected in 8 of 39 samples was demonstrated to be consistent with an artifact of plasmid-derived SV40. In contrast to recent reports, we obtained substantial quantitative evidence indicating that neither HPV nor any other pRb-inactivating human DNA tumor viruses play a role in the development of retinoblastoma, regardless of RB1 genotype.

Child↗
PubMed2007

Profiling genomic copy number changes in retinoblastoma beyond loss of RB1.

Loss of both RB1 alleles is rate limiting for development of retinoblastoma (RB), but genomic copy number gain or loss may impact oncogene(s) and tumor suppressor genes, facilitating tumor progression. We used quantitative multiplex polymerase chain reaction to profile "hot spot" genomic copy number changes for gain at 1q32.1, 6p22, and MYCN, and loss at 16q22 in 87 primary RB and 7 cell lines. Loss at 16q22 (48%) negatively associated with MYCN gain (18%) (Fisher's exact P = 0.031), gain at 1q32.1 (62%) positively associated with 6p "hot spot" gain (43%) (P = 0.033), and there was a trend for positive association between 1q and MYCN gain (P = 0.095). Cell lines had a higher frequency of MYCN amplification than primary tumors (29% versus 3%; P = 0.043). Novel high-level amplification of 1q32.1 in one primary tumor, confirmed by fluorescence in situ hybridization, strongly supports the presence of oncogene(s) in this region, possibly the mitotic kinesin, KIF14. Gene-specific quantitative multiplex polymerase chain reaction of candidate oncogenes at 1q32.1 (KIF14), 6p22 (E2F3 and DEK), and tumor suppressor genes at 16q22 (CDH11) and 17q21 (NGFR) showed the most common gene gains in RB to be KIF14 in cell lines (80%) and E2F3 in primary tumors (70%). The patterns of gain/loss were qualitatively different in 25 RB compared with 12 primary hepatocellular carcinoma and 12 breast cancer cell lines. Gene specific analysis of one bone marrow metastasis of RB, prechemotherapy and postchemotherapy, showed the typical genomic changes of RB pretreatment, which normalized after chemotherapy.

Base Sequence↗
PubMed2006

KIF14 mRNA expression is a predictor of grade and outcome in breast cancer.

Gain of chromosome 1q is a hallmark of breast cancer, and likely reflects oncogene amplification. We previously identified mitotic kinesin KIF14 (kinesin family member 14) as an overexpressed candidate oncogene in the 1q31.3-1q32.1 minimal region of genomic gain in breast cancer cell lines. KIF14 also showed high expression in other cancers, notably an association with survival in lung tumors. We now report KIF14 expression in 99 primary breast tumors and 10 normal breast controls. Measured by real-time RT-PCR, KIF14 was overexpressed 10-fold on average in tumors relative to normals (t test p = 0.000054); expression increased with grade (ANOVA p = 0.000006). Infiltrating ductal carcinomas had higher KIF14 levels than lobular (p = 0.017), and estrogen receptor (ER) negative tumors had higher KIF14 levels than ER positive tumors (t test p = 0.030). KIF14 expression correlated positively with Ki-67 mRNA level (Spearman r = 0.692, p = 0.000001), fraction of positive nodes (r = 0.227, p = 0.024) and percent invasive cells (r = 0.360, p = 0.0002), and negatively with percent fatty stroma (r = -0.258, p = 0.010) and percent normal epithelium (r = -0.291, p = 0.003). KIF14 expression is thus tumor-specific and increased in more aggressive tumors. Indeed, KIF14 expression predicted overall survival (univariate Cox p = 0.010), with an odds ratio of 3.60 (1.37-9.48), in 50 tumors with available outcome data. KIF14 overexpression also predicted decreased disease-free survival (log-rank p = 0.049). These findings are the first evidence of association between expression of a mitotic kinesin and prognostic variables in breast cancer.

Adult↗
PubMed2006

Expression analysis of 6p22 genomic gain in retinoblastoma.

To identify gene(s) targeted by 6p22 genomic gain, present in more than 50% retinoblastoma tumors, we used real-time RT-PCR to quantify the expression of seven genes in normal human retina and retinoblastoma. Six genes are located in the quantitative multiplex PCR-defined 0.6 Mb minimal region of gain at 6p22 (DEK, AOF1, TPMT, NHLRC1, KIF13A, and NUP153), and E2F3 is 2 Mb away from the minimal region of gain on 6p22. E2F3, DEK, KIF13A, and NUP153 were most frequently overexpressed in retinoblastoma with 6p genomic gain, compared with the normal adult human retina. E2F3 and DEK mRNA levels were increased in all human tumors showing 6p22 gain, as well as in mouse retinoblastoma induced by SV40 large T antigen expression in developing retina, compared with the normal controls (adult human retina and 7-day-old mouse retina, respectively). Only DEK showed statistically significant correlation of expression and genomic copy number (P = 0.019). E2F3 and DEK, but not NUP153, showed developmental regulation. E2F3 and DEK mRNA overexpression was always associated with protein overexpression, determined by immunoblotting or immunofluorescent staining of primary tumors, relative to the adjacent normal retina. E2F3 was strongly expressed in actively proliferating cells, while DEK was overexpressed in all tumor cells. Taking into account the proliferation-promoting role of E2F3, implication of E2F3 in bladder and prostate cancer, and the translocation and overexpression of DEK in leukemia, we conclude that either DEK or E2F3 (or both) are targeted by the 6p22 gain in retinoblastoma.

Adult↗
PubMed2005

KIF14 is a candidate oncogene in the 1q minimal region of genomic gain in multiple cancers.

Gain of chromosome 1q31-1q32 is seen in >50% of retinoblastoma and is common in other tumors. To define the minimal 1q region of gain, we determined genomic copy number by quantitative multiplex PCR of 14 sequence tagged sites (STSs) spanning 1q25.3-1q41. The most frequently gained STS at 1q32.1 (71%; 39 of 55 retinoblastoma) defined a 3.06 Mbp minimal region of gain between flanking markers, containing 14 genes. Of these, only KIF14, a putative chromokinesin, was overexpressed in various cancers by real-time RT-PCR. KIF14 mRNA was expressed in 20/22 retinoblastoma samples 100-1000-fold higher than in retina (t-test P=0.00002); cell lines (n=10) had higher levels than tumors (n=12) (P=0.009). KIF14 protein was overexpressed in retinoblastoma tumors and breast cancer cell lines by immunoblot. KIF14 was expressed in 4/4 breast cancer cell lines 31-92-fold higher than in normal breast tissue, in 5/5 medulloblastoma cell lines 22-79-fold higher than in fetal brain, and in 10/22 primary lung tumors 3-34-fold higher than in normal lung. Patients with lung tumors that overexpress KIF14 showed a trend toward decreased survival. KIF14 may thus be important in oncogenesis, and has promise as a prognostic indicator and therapeutic target.

Breast Neoplasms↗
PubMed2005

Distinct patterns of expression of the RB gene family in mouse and human retina.

Although RB1 function is disrupted in the majority of human cancers, an undefined cell of developing human retina is uniquely sensitive to cancer induction when the RB1 tumor suppressor gene is lost. Murine retinoblastoma is initiated only when two of the RB family of genes, RB1 and p107 or p130, are inactivated. Although whole embryonic retina shows RB family gene expression by several techniques, when E14 developing retina was depleted of the earliest differentiating cells, ganglion cells, the remaining proliferating murine embryonic retinal progenitor cells clearly did not express RB1 or p130, while the longer splice form of p107 was expressed. Each retinal cell type expressed some member of the RB family at some stage of differentiation. Rod photoreceptors stained for the RB1 protein product, pRB, and p107 in only a brief window of postnatal murine development, with no detectable staining for any of the RB family proteins in adult human and mouse rod photoreceptors. Adult mouse and human Muller glia, ganglion and rare horizontal cells, and adult human, but not adult mouse, cone photoreceptors stained for pRB. The RB gene family is dynamically and variably expressed through retinal development in specific retinal cells.

Animals↗
PubMed2005

Chemotherapy for retinoblastoma.

Retinoblastoma is the most common eye cancer in children. Pilot studies of chemotherapy for intraocular retinoblastoma have been reported by several groups, using different combinations, dosages, schedules, and durations of carboplatin, etoposide, or teniposide, with or without vincristine, and with or without cyclosporine to counteract multidrug resistance. All studies of chemotherapy for intraocular retinoblastoma have included consolidation by focal therapy, with or without radiation. Chemotherapy alone reduces tumor size but does not cure retinoblastoma. Focal therapy, consisting of photocoagulation, thermotherapy, cryotherapy, or brachytherapy, is necessary to consolidate chemotherapy response.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2004

Retinoblastoma: revisiting the model prototype of inherited cancer.

Hereditary retinoblastoma is an autosomal dominant disorder caused by mutations in the RB1 gene. Analysis of this rare condition has helped to elucidate the mechanisms underlying hereditary cancer predisposition in general. As identification of RB1 gene mutations has become a part of clinical management of patients with retinoblastoma, there is now a wealth of data. In this article, we summarize the current knowledge on the relations between the genotype and phenotypic expression. Moreover, detailed analysis of genotype-phenotype relations shows that hereditary retinoblastoma has features of a complex trait.

Child, Preschool↗
PubMed2004

Defining a 0.5-mb region of genomic gain on chromosome 6p22 in bladder cancer by quantitative-multiplex polymerase chain reaction.

Metaphase-based comparative genomic hybridization (CGH) has identified recurrent regions of gain on different chromosomes in bladder cancer, including 6p22. These regions may contain activated oncogenes important in disease progression. Using quantitative multiplex polymerase chain reaction (QM-PCR) to study DNA from 59 bladder tumors, we precisely mapped the focal region of genomic gain on 6p22. The marker STS-X64229 had copy number increases in 38 of 59 (64%) tumors and the flanking markers, RH122450 and A009N14, had copy number gains in 33 of 59 (56%) and 26 of 59 (45%) respectively. Contiguous gain was present for all three markers in 14 of 59 (24%) and for two (RH122450 and STS-X64229) in 25 of 59 (42%). The genomic distance between the markers flanking STS-X64229 is 0.5 megabases, defining the minimal region of gain on 6p22. Locus-specific interphase fluorescence in situ hybridization confirmed the increased copy numbers detected by QM-PCR. Current human genomic mapping data indicates that an oncogene, DEK, is centrally placed within this minimal region. Our findings demonstrate the power of QM-PCR to narrow the regions identified by CGH to facilitate identifying specific candidate oncogenes. This also represents the first study identifying DNA copy number increases for DEK in bladder cancer.

Adult↗
PubMed2004

Retinoblastoma protein purification and transduction of retina and retinoblastoma cells using improved alphavirus vectors.

PURPOSE: To develop and use improved Semliki Forest vectors (SFVs) for functional and structural analyses of the retinoblastoma protein (RB) in developing retina and retinoblastoma cells. METHODS: Virus was harvested from cells transfected with replicon and helper plasmids. Combinations of producer and target cells were tested for optimal virus production and protein expression. The replicon was improved by adding an expanded multiple cloning site, translational enhancer, and FLAG and HIS10 epitope and affinity tags. Affinity chromatography was used to purify beta-galactosidase or RB. RB function was assessed through interaction with E2F1. The efficacy of SFV as a retinal delivery system was tested on mouse explants and cultured human retinoblastoma cells. RESULTS: The optimal producer and target cell lines were an HEK-293 derivative (Phoenix Eco) and BHK-21, respectively. Stable expression of structural proteins in the BHK-21 helper line simplified virus production and amplified virus yield 100-fold. The translational enhancer improved expression three- to eightfold. Full-length, functional RB was produced without the truncated products characteristic of bacterially produced RB and was purified using the affinity tags. SFVs efficiently transduced mouse retinal explants and multiple hard-to-transduce retinoblastoma tumor lines. CONCLUSIONS: This study describes a simple, rapid, SFV vector system to produce recombinant proteins, such as RB, in mammalian cells. These SFV vectors represent an efficient approach to purification of proteins and protein complexes and transduction of retinal or retinoblastoma cells for the purpose of in vivo analysis of protein functions.

Alphavirus↗
PubMed2004

Minimal 16q genomic loss implicates cadherin-11 in retinoblastoma.

Retinoblastoma is initiated by loss of both RB1 alleles. Previous studies have shown that retinoblastoma tumors also show further genomic gains and losses. We now define a 2.62 Mbp minimal region of genomic loss of chromosome 16q22, which is likely to contain tumor suppressor gene(s), in 76 retinoblastoma tumors, using loss of heterozygosity (30 of 76 tumors) and quantitative multiplex PCR (71 of 76 tumors). The sequence-tagged site WI-5835 within intron 2 of the cadherin-11 (CDH11) gene showed the highest frequency of loss (54%, 22 of 41 samples tested). A second hotspot for loss (39%, 9 of 23 samples tested) was detected within intron 2 of the cadherin-13 (CDH13) gene. Furthermore, deletion of the exons of CDH11 and/or WI-5835 was shown by quantitative multiplex PCR in 17 of 30 (57%) of previously untested tumors. Immunoblot analyses revealed that 91% (20 of 22) retinoblastoma exhibited either a complete loss or a decrease of the intact form of CDH11 and 8 of 13 showed a prevalent band suggestive of the variant form. Copy number of WI-5835 for these samples correlated with CDH11 protein expression. CDH11 staining was evident in the inner nuclear layer in early mouse retinal development and in small transgenic murine SV40 large T antigen-induced retinoblastoma tumors, but advanced tumors frequently showed loss of CDH11 expression by reverse transcription-PCR, suggestive of a role for CDH11 in tumor progression or metastasis. CDH13 protein and mRNA were consistently expressed in all human and murine retinoblastoma compared with normal adult human retina. Our analyses implicate CDH11, but not CDH13, as a potential tumor suppressor gene in retinoblastoma.

Animals↗
PubMed2003

Unique insertional translocation in a childhood Wilms' tumor survivor detected when his daughter developed bilateral retinoblastoma.

Retinoblastoma and Wilms' tumor are rare childhood embryonic tumors associated with loss or inactivation of tumor suppressor genes, RB1 located within 13q14, and WT1 located within 11p13. Interchromosomal insertional translocations occur rarely, and such rearrangements within RB1 or WT1, even rarer. We report a unique family in which an insertional translocation of a chromosomal segment that included band 13q14 inserted into 11p13 caused childhood Wilms' tumor in the father, and whose child developed bilateral retinoblastoma. This is the first case of an insertional translocation that caused both tumors. This insertional translocation had significant consequences for genetic counseling and in utero diagnosis. The estimated risk for an offspring of this father to develop Wilms' tumor is up to 50%, to develop retinoblastoma up to 25%, to have neither tumor 25%, and to have both tumors 0%.

Chromosome Banding↗
PubMed2003

Ocular motility changes after subtenon carboplatin chemotherapy for retinoblastoma.

BACKGROUND: Focal subtenon carboplatin injections have recently been used as a presumably toxicity-free adjunct to systemic chemotherapy for intraocular retinoblastoma. OBJECTIVE: To report our clinical experience with abnormal ocular motility in patients treated with subtenon carboplatin chemotherapy. METHODS: We noted abnormal ocular motility in 10 consecutive patients with retinoblastoma who had received subtenon carboplatin. During ocular manipulation under general anesthesia, we assessed their eyes by forced duction testing, comparing ocular motility after tumor control with ocular motility at diagnosis. Eyes subsequently enucleated because of treatment failure (n = 4) were examined histologically. RESULTS: Limitation of ocular motility was detected in all 12 eyes of 10 patients treated for intraocular retinoblastoma with 1 to 6 injections of subtenon carboplatin as part of multimodality therapy. Histopathological examination revealed many lipophages in the periorbital fat surrounding the optic nerve in 1 eye, indicative of phagocytosis of previously existing fat cells and suggesting prior fat necrosis. The enucleations were technically difficult and hazardous for globe rupture because of extensive orbital soft tissue adhesions. CONCLUSIONS: Subtenon carboplatin chemotherapy is associated with significant fibrosis of orbital soft tissues, leading to mechanical restriction of eye movements and making subsequent enucleation difficult. Subtenon carboplatin is not free of toxicity, and its use is best restricted to specific indications.

Adolescent↗
PubMed2002

Genomic amplification in retinoblastoma narrowed to 0.6 megabase on chromosome 6p containing a kinesin-like gene, RBKIN.

All retinoblastomas (RBs) show genomic changes in addition to loss of both RB1 alleles. Quantitative-multiplex PCR analysis identified in 41 of 70 (59%) RBs a 0.6-Mb minimal region of chromosome 6p22 gain from which we cloned the human kinesin gene, RBKIN/KIF13A, by rapid amplification of retinal cDNA. RBKIN is expressed ubiquitously in adult tissues, at low levels in fetal tissues, and at high levels in RB. Antisense RBKIN oligonucleotide reduced the growth rate of RB cell lines. RBKIN and/or another closely linked gene are candidate oncogenes contributing to malignant progression of RB.

Cell Division↗
PubMed2002

Foveal and eccentric acuity in one-eyed observers.

The purpose of these two experiments was to examine the effects of enucleation on the foveal and near-periphery acuity of human observers. In Experiment 1, using high contrast illiterate-E optotypes, we measured foveal and 7-degree eccentric (nasal and temporal) acuity in the remaining eye of unilaterally enucleated children and adults. The data were compared to those of the fellow or 'good eye' of age-matched non-amblyopic and amblyopic strabismic observers-a different kind of monocular deprivation-and binocularly normal subjects. The main finding was that the 7-degree eccentric acuity of enucleated observers and normal controls was best, followed by that of the non-amblyopic and lastly, the amblyopic subjects. There were no naso-temporal asymmetries. In Experiment 2, foveal and 7-degree eccentric acuities of enucleated and binocularly normal subjects were tested at 96, 13.5 and 4.7% contrast levels using the same kind of stimuli. The foveal acuity of the enucleated observers was better than the monocular acuity of controls at all contrasts, and equivalent to their binocular acuity. The eccentric acuity of the enucleated observers was better at the two low contrast levels. No naso-temporal asymmetries were found in either group at any contrast level. These findings suggest that, in humans, visual deprivation from unilateral enucleation does not result in a naso-temporal asymmetry in the near periphery.

Adolescent↗