Keith Collins - Academia.edu (original) (raw)

Papers by Keith Collins

The EMBO Journal, 2001

Cyclin A-mediated activation of cyclin-dependent kinases (CDKs) is essential for cell cycle trans... more Cyclin A-mediated activation of cyclin-dependent kinases (CDKs) is essential for cell cycle transversal. Cyclin A activity is regulated on several levels and cyclin A elevation in a number of cancers suggests a role in tumorigenesis. In the present study, we used a modi®ed DNA binding site selection and PCR ampli®cation procedure to identify DNA binding proteins that are potential substrates of cyclin A±CDK. One of the sequences identi®ed is the Sp1 transcription factor binding site. Co-immunoprecipitation experiments show that cyclin A and Sp1 can interact physically. In vitro and in vivo phosphorylation studies indicate that cyclin A±CDK complexes can phosphorylate Sp1. The phosphorylation site is located in the N-terminal region of the protein. Cells overexpressing cyclin A have elevated levels of Sp1 DNA binding activity, suggesting that cyclin A±CDK-mediated phosphorylation augments Sp1 DNA binding properties. In co-transfection studies, cyclin A expression stimulated transcription from an Sp1-regulated promoter. Mutation of the phosphorylation site abrogated cyclin A±CDKdependent phosphorylation, augmentation of Sp1 transactivation function and DNA binding activity.

Cancer research, Jan 15, 1994

An allelotype analysis of endometrial carcinoma was undertaken to identify chromosomal loci that ... more An allelotype analysis of endometrial carcinoma was undertaken to identify chromosomal loci that are relevant to this tumor type. A total of 70 highly polymorphic microsatellite markers, distributed among all nonacrocentric chromosome arms, were examined for evidence of loss of heterozygosity or allelic imbalance in DNA samples from matched normal and tumor tissues. An average of 21 informative tumor cases were obtained for each marker. Allelic deletions or imbalance were observed on 31 of 41 chromosome arms with no marker showing an allelic loss ratio of greater than 33%. Those chromosome arms most frequently involved were 3p, 8p, 9p, 14q, 16q and 18q. There was a strong correlation between loss of heterozygosity on chromosome 14q and death from disease. These data indicate that the molecular genetic character of endometrial carcinoma is complex and that a relatively large number of different chromosomal loci are likely to play a role in the etiology and progression of this tumor t...

Toxicologic Pathology, 2006

Mutations in both p53 and BRCA2 are commonly seen together in human tumors suggesting that the lo... more Mutations in both p53 and BRCA2 are commonly seen together in human tumors suggesting that the loss of both genes enhances tumor development. To elucidate this interaction in an animal model, mice lacking the carboxy terminal domain of Brca2 were crossed with p53 heterozygous mice. Females from this intercross were then irradiated with an acute dose of 5 Gy ionizing radiation at 5 weeks of age and compared to nonirradiated controls. We found decreased survival and timing of tumor onsets, and significantly higher overall tumor incidences and prevalence of particular tumors, including stomach tumors and squamous cell carcinomas, associated with the homozygous loss of Brca2, independent of p53 status. The addition of a p53 mutation had a further impact on overall survival, incidence of osteosarcomas and stomach tumors, and tumor latency. The spectrum of tumors observed for this Brca2 germline mouse model suggest that it faithfully recapitulates some human disease phenotypes associated ...

Human Molecular Genetics, 2005

The most common cause of primary autosomal recessive microcephaly (MCPH) appears to be mutations ... more The most common cause of primary autosomal recessive microcephaly (MCPH) appears to be mutations in the ASPM gene which is involved in the regulation of neurogenesis. The predicted gene product contains two putative N-terminal calponin-homology (CH) domains and a block of putative calmodulin-binding IQ domains common in actin binding cytoskeletal and signaling proteins. Previous studies in mouse suggest that ASPM is preferentially expressed in the developing brain. Our analyses reveal that ASPM is widely expressed in fetal and adult tissues and upregulated in malignant cells. Several alternatively spliced variants encoding putative ASPM isoforms with different numbers of IQ motifs were identified. The major ASPM transcript contains 81 IQ domains, most of which are organized into a higher order repeat (HOR) structure. Another prominent spliced form contains an in-frame deletion of exon 18 and encodes 14 IQ domains not organized into a HOR. This variant is conserved in mouse. Other spliced variants lacking both CH domains and a part of the IQ motifs were also detected, suggesting the existence of isoforms with potentially different functions. To elucidate the biochemical function of human ASPM, we developed peptide specific antibodies to the N-and C-termini of ASPM. In a western analysis of proteins from cultured human and mouse cells, the antibodies detected bands with mobilities corresponding to the predicted ASPM isoforms. Immunostaining of cultured human cells with antibodies revealed that ASPM is localized in the spindle poles during mitosis. This finding suggests that MCPH is the consequence of an impairment in mitotic spindle regulation in cortical progenitors due to mutations in ASPM.

Toxins, 2018

LMB-100 is a mesothelin-targeted recombinant immunotoxin (iTox) that carries a modified Pseuodomo... more LMB-100 is a mesothelin-targeted recombinant immunotoxin (iTox) that carries a modified Pseuodomonas exotoxin A (PE) payload. PE kills cells by inhibiting synthesis of new proteins. We found that treatment of pancreatic cancer cells with LMB-100 for 24–48 h did not change total protein level despite inducing protein synthesis inhibition (PSI). Further, increased levels of ubiquitinated proteins were detected, indicating that cells may have limited ability to compensate for PSI by reducing protein degradation. Together, these data suggest that PE depletes concentrations of a minority of cellular proteins. We used reverse phase protein array and Luminex assay to characterize this subset. LMB-100 decreased the abundance of 24 of 32 cancer-related proteins (including Bcl-x, Her2, Her3 and MUC16) without compensatory increases in other analytes. Further, cancer cells failed to maintain extracellular concentrations of cancer cell secreted growth factors (CCSGFs), including Vascular Endoth...

Journal of Complementary and Integrative Medicine, 2011

Cancer research, 2000

Women who inherit mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2, are predi... more Women who inherit mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2, are predisposed to the development of breast and ovarian cancer. We used mice with a Brca1 mutation on a BALB/cJ inbred background (BALB/cB1+/- mice) or a Brca2 genetic alteration on the 129/SvEv genetic background (129B2+/- mice) to investigate potential gene-environment interactions between defects in these genes and treatment with the highly estrogenic compound diethylstilbestrol (DES). Beginning at 3 weeks of age, BALB/cB1+/-, 129B2+/-, and wild-type female mice were fed a control diet or a diet containing 640 ppb DES for 26 weeks. DES treatment caused vaginal epithelial hyperplasia and hyperkeratosis, uterine inflammation, adenomyosis, and fibrosis, as well as oviductal smooth muscle hypertrophy. The severity of the DES response was mouse strain specific. The estrogen-responsive 129/SvEv strain exhibited an extreme response in the reproductive tract, whereas the effect in BALB/cJ and C3H/HeN...

Breast Cancer Research

While advances in science and technology have increased options for treating breast cancer, curre... more While advances in science and technology have increased options for treating breast cancer, current social trends have changed the way people deal with this disease. Women in the United States are no longer simply passive patients, but rather they are survivors, advocates and activists who are speaking up for themselves and speaking out for issues relevant to the treatment and prevention of breast cancer. As the discoveries of basic science have been translated to better clinical treatment, a new sense of hope has emerged. Quality of life now shares the spotlight with quantity of life as breast cancer has shifted from an acute to a chronic condition and as the numbers of long-term survivors increase. While this new population tends to have more optimistic expectations for survival, they are also expressing concerns about issues affecting their lives through and beyond treatment. These issues include, but are not limited to, such concerns as efficient and accurate diagnosis, the complexity of treatment decisions, access to quality cancer care, informed consent, privacy issues, availability of supportive care treatments, and effective communication skills, especially with their physicians. Survivors are also concerned about the impact of their disease on spouses and family, on fertility and sexuality issues, on their employment and (in the USA) insurability, and on their long-term survival. The identification of these increasing issues has given rise to a consumer movement that encourages a shift away from powerless victim to empowered survivor.

Molecular Cancer Therapeutics, 2015

p53 is a well-characterized transcription factor that is mutated in about 50% of human cancers. T... more p53 is a well-characterized transcription factor that is mutated in about 50% of human cancers. The majority of p53 genomic alterations are missense mutations which result in expression of p53 protein isoforms with deficient functionality. Such missense mutations cluster in several hotspots, with the most frequent alterations located at amino acids 175, 248 and 273 of human p53 (mouse amino acids 172, 245 and 270). p53 missense mutants disrupt the function of p53 tetramers, thereby behaving similarly to loss of p53. However, there is evidence that p53 missense mutants have additional tumor-promoting functions that differ from p53 deletions: p53 missense mutants can transform p53 null cells in vitro and in mouse models, and the p53 missense mutants induce a different tumor spectrum from that induced by p53 loss or other p53 missense mutants. The information obtained from the engineered murine models featuring p53 missense mutations has greatly elucidated the role of p53 mutagenesis in cancer. However, the currently-available p53 models bear several drawbacks that make them sub-optimal for preclinical studies. These models contain a global p53 haploinsufficiency, potentially impacting the biology of tumor-modifying structures like stroma, the immune system, and the vasculature. Furthermore, activation of the p53 missense mutant alleles results in a transition from a single functional wild-type allele to a combination of a wild-type and a mutant allele. These features substantially deviate from those of observed in human tumorigenesis, where somatic p53 mutagenesis in cells undergoing transformation results in a transition from two wild-type alleles to a combination of a wild-type and a mutant alleles, while tumor-modifying structures retain an unaltered p53 biallelic configuration. Here we describe the molecular and phenotypic characterization of a new allelic series of conditional p53 missense mutant mouse lines in which Cre-mediated recombination converts p53 from a wild-type to a missense mutant (R172H, R270H or R270C) configuration. To characterize the gradual molecular changes induced by expression of R172H and R270H mutants, we derived MEF lines harboring the alleles in a heterozygous conditional (p53-R172H fl/+ or p53-R270H fl/+) configuration. We observed that the wild-type p53 mRNA was lost but p53 protein abundance was increased after Cre-mediated activation of the mutant allele. Interestingly, we found that the R270H mutant allele provided a greater proliferative advantage, a distinct growth pattern and a greater ability to grow in vitro under starvation conditions than a similarly engineered R172H mutant allele. Consistent with published evidence, these observations suggest that the acquired functional changes are dissimilar between different p53 missense mutants. We further characterized the transcriptome profiles in MEF lines harboring R172H and R270H alleles and identified a number of unique and common transcriptional changes that could be causal for gain-of-function phenotypes and informative for uncovering additional mechanisms for carcinogenesis driven by p53 missense mutants. We used the newly-derived p53 mutant alleles to establish improved autochthonous and orthotopic pancreatic cancer models suitable for preclinical efficacy studies and we are currently developing lung cancer, ovarian cancer, and medullary thyroid carcinoma models. Citation Format: Tomas Vilimas, Keith Collins, Theresa Guerin, Roackie Awasthi, Lionel Feigenbaum, Guillermina Lozano, Terry Van Dyke, Serguei Kozlov. p53 missense mutants R172H and R270H exhibit differential effects on tumorigenesis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A17.

Journal of Complementary Integrative Medicine, 2011

Women who are diagnosed with breast cancer often self-administer complementary and alternative me... more Women who are diagnosed with breast cancer often self-administer complementary and alternative medicines to augment their conventional treatments, improve health, or prevent recurrence. Flor-Essence® herbal tonic is a complex mixture of eight herbal extracts used by cancer patients because of anecdotal evidence that it can treat or prevent disease. In this study four experimental groups of female MMTV-Neu mice were left untreated or treated with 3% Flor-Essence® in utero, from birth until 5 weeks of age, or throughout their lifetime. Palpable mammary tumor incidence and body weight was determined weekly for each group. The mice were sacrificed at 28 weeks of age and mammary tumors were enumerated to determine average tumor incidence and multiplicity for each group. Female mice exposed to Flor-Essence® herbal tonic in utero weighed significantly more than the control group (p < 0.001). The average tumor incidence and tumor multiplicity in the experimental mice treated with Flor-Essence® herbal tonic did not differ from the control animals. Flor-Essence® does not inhibit mammary tumor incidence or mammary tumor multiplicity in MMTV-Neu transgenic mice. Flor-Essence® exposure in utero causes increased body weight in experimental animals. This conclusion challenges widely available anecdotal information as well as the hopes of the consumer that this product will inhibit or suppress tumor development.Lay AbstractFlor-Essence® herbal tonic is a complex mixture of eight herbal extracts often used by women with breast cancer in hopes that it will help cure disease or prevent recurrence. There is currently very little scientific data to support or refute its self-administration. We tested whether Flor-Essence® would influence tumor development in the mammary glands of a mouse model of Her2/neu breast cancer. The tonic was given at different life stages to determine if timing of the exposure influenced the response to treatment. This report shows that Flor-Essence® did not inhibit mammary tumor development in the treated mice challenging anecdotal information, as well as the hopes of the consumer, that this product will inhibit or suppress tumor development. This report also shows that Flor-Essence® exposure in utero causes increased body weight in experimental animals.

Toxicologic pathology

Inherited BRCA2 mutations predispose individuals to breast cancer and increase risk at other site... more Inherited BRCA2 mutations predispose individuals to breast cancer and increase risk at other sites. Recent studies have suggested a role for the APC I1307K allele as a low-penetrance breast cancer susceptibility gene that enhances the phenotypic effects of BRCA1 and BRCA2 mutations. To model the consequences of inheriting mutant alleles of the BRCA2 and APC tumor suppressor genes, we examined tumor outcome in C57BL/6 mice with mutations in the Brca2 and Apc genes. We hypothesized that if the Brca2 and Apc genes were interacting to influence mammary tumor susceptibility, then mammary tumor incidence and/or multiplicity would be altered in mice that had inherited mutations in both genes. Female and male offspring treated with a single IP injection of 50 mg/kg N-ethyl-N-nitrosourea (ENU) at 35 days of age developed mammary adenoacanthomas by 100 days of age. The female Apc-mutant and Brca2/Apc double-mutant progeny had mean mammary tumor multiplicities of 6.7+/-2.8 and 7.2+/-2.7, respe...

Proceedings of the National Academy of Sciences, 2004

Molecular Carcinogenesis, 2000

Women who inherit mutations in the BRCA2 cancer susceptibility gene have an 85% chance of develop... more Women who inherit mutations in the BRCA2 cancer susceptibility gene have an 85% chance of developing breast cancer. The function of the BRCA2 gene remains elusive, but there is evidence to support its role in transcriptional transactivation, tumor suppression, and the maintenance of genomic integrity. Individuals with identical BRCA2 mutations display a different distribution of cancers, suggesting that there are low-penetrance genes that can modify disease outcome. We hypothesized that genetic background could influence embryonic survival of a Brca2 mutation in mice. Brca2-null embryos with a 129/SvEv genetic background (129(B2-/-)) died before embryonic day 8. 5. Transfer of this Brca2 mutation onto the BALB/cJ genetic background (BALB/c(B2-/-)) extended survival to embryonic day 10.5. These results indicate that the BALB/c background harbors genetic modifiers that can prolong Brca2-null embryonic survival. The extended survival of BALB/c(B2-/-) embryos enabled us to ask whether transcriptional regulation of the Brca1 and Brca2 genes is interdependent. The interdependence of Brca1 and Brca2 was evaluated by studying Brca2 gene expression in BALB/c(B1-/-) embryos and Brca1 gene expression in BALB/c(B2-/-) embryos. Nonisotopic in situ hybridization demonstrated that Brca2 transcript levels were comparable in BALB/c(B1-/-) embryos and wild-type littermates. Likewise, reverse transcriptase-polymerase chain reactions confirmed Brca1 mRNA expression in embryonic day 8.5 BALB/c(B2-/-) embryos that was comparable to Brca2-heterozygous littermates. Thus, the Brca1 and Brca2 transcripts are expressed independently of one another in Brca1- and Brca2-null embryos. Mol. Carcinog. 28:174-183, 2000.

Toxicologic …, 2001

Molecular …, 2000