Biomedical subjects

Gary A Clawson

Publications and source records attributed to Gary A Clawson.

13 recordsLinked to original sources

PubMed2007

The chloromethylketone protease inhibitor AAPF(CMK) also targets ATP-dependent helicases and SAP-domain proteins.

We have been studying a nuclear protease, which appears to be involved in cellular transformation, as well as in infections with high-risk human papillomaviruses (HPVs). This protease has a chymotrypsin-like substrate specificity and the chloromethylketone inhibitor AAPF(CMK) is a potent (and relatively selective) inhibitor of it. Recently, we have observed that AAPF(CMK) has potent effects in some model systems which appear not to be mediated by decreases in the nuclear protease. Here we show that AAPF(CMK) selectively reacts with ATP-dependent helicases as well as a limited spectrum of proteins in other DNA repair/chromatin remodeling nuclear complexes, including for example Cohesin complex components and proteins containing SAP-domains. In vitro, AAPF(CMK) selectively reacts with SV40 large T antigen, and inhibits its helicase activity.

Adenosine Triphosphate↗

PubMed2006

Antisense applications for biological control.

Although Nature's antisense approaches are clearly impressive, this Perspectives article focuses on the experimental uses of antisense reagents (ASRs) for control of biological processes. ASRs comprise antisense oligonucleotides (ASOs), and their catalytically active counterparts ribozymes and DNAzymes, as well as small interfering RNAs (siRNAs). ASOs and ribozymes/DNAzymes target RNA molecules on the basis of Watson-Crick base pairing in sequence-specific manner. ASOs generally result in destruction of the target RNA by RNase-H mediated mechanisms, although they may also sterically block translation, also resulting in loss of protein production. Ribozymes and DNAzymes cleave target RNAs after base pairing via their antisense flanking arms. siRNAs, which contain both sense and antisense regions from a target RNA, can mediate target RNA destruction via RNAi and the RISC, although they can also function at the transcriptional level. A considerable number of ASRs (mostly ASOs) have progressed into clinical trials, although most have relatively long histories in Phase I/II settings. Clinical trial results are surprisingly difficult to find, although few ASRs appear to have yet established efficacy in Phase III levels. Evolution of ASRs has included: (a) Modifications to ASOs to render them nuclease resistant, with analogous modifications to siRNAs being developed; and (b) Development of strategies to select optimal sites for targeting. Perhaps the biggest barrier to effective therapies with ASRs is the "Delivery Problem." Various liposomal vehicles have been used for systemic delivery with some success, and recent modifications appear to enhance systemic delivery, at least to liver. Various nanoparticle formulations are now being developed which may also enhance delivery. Going forward, topical applications of ASRs would seem to have the best chances for success. In summary, modifications to ASRs to enhance stability, improve targeting, and incremental improvements in delivery vehicles continue to make ASRs attractive as molecular therapeutics, but their advance toward the bedside has been agonizingly slow.

Animals↗

PubMed2006

A protease inhibitor specifically inhibits growth of HPV-infected keratinocytes.

Protease inhibitors have been known to exhibit anticarcinogenic activity in a variety of model systems, although the biological target(s) and mechanism remain enigmatic. Human papillomavirus (HPV) is the primary etiological agent of cervical cancer. Here we show that a nuclear chymotrypsin-like protease activity (NCLPA), which appears to be involved in transformation in several different experimental models, is significantly elevated in keratinocytes infected with high-risk HPV. Further, we demonstrate a marked growth inhibition of organotypic raft cultures, which is specific for cells infected with high-risk HPV types, using a chloromethyl ketone inhibitor previously shown to be relatively selective for the NCLPA. Surprisingly, this HPV-dependent inhibitory effect is independent of any alterations in the NCLPA. This finding has clear implications for the development of novel therapeutics specifically targeted to cervical dysplasias with HPV-infected cells.

Amino Acid Chloromethyl Ketones↗

PubMed2006

Insulin-like growth factor-1 induces lipid production in human SEB-1 sebocytes via sterol response element-binding protein-1.

An understanding of the molecular signaling involved in sebaceous gland lipid production is needed to develop therapeutic targets to improve acne. Treatment with methylisobutylxanthine, dexamethasone, and a high dose of insulin (MDI) has been shown to differentiate 3T3-L1 preadipocytes into adipocytes, a differentiation marked by an increase in lipid production. The present study has the following aims: (1) Since high doses of insulin, as found in MDI, will activate the IGF-1 receptor, we sought to determine if IGF-1 is capable of reproducing the lipogenic effect seen with MDI treatment, and (2) to determine if the sterol response element-binding protein-1 (SREBP-1) pathway mediates the increase in lipogenesis. Here we report that MDI increases lipogenesis and that this effect can be attributed wholly to the high-dose insulin in SEB-1 cells. Further, we show that a physiologically relevant dose of IGF-1 or high-dose (1 microM) insulin induces an increase in SREBP-1 mRNA, protein, and total lipid production; while 100 nM insulin induces lipogenesis yet the SREBP protein levels remain unchanged. These data indicate that activation of the IGF-1 receptor increases lipogenesis in SEB-1 cells through both SREBP-dependent and SREBP-independent pathways.

Cells, Cultured↗

PubMed2006

Identifying accessible sites in RNA: the first step in designing antisense reagents.

There is continued interest in development of antisense reagents (ASRs), including especially antisense oligonucleotides and small interfering RNAs, for experimental as well as therapeutic purposes. Optimization of ASRs begins with target site selection. Here we review protocols which have been developed to empirically determine effective target sites in RNAs. Such library selection technologies have demonstrated clear utility, and in vitro identification of sites has generally proven effective for cellular applications. A few groups are developing large combinatorial libraries and approaches to adapt use of such libraries to individual target RNAs, as well as learning algorithms to help with the optimization of target sites, particularly with respect to small interfering RNAs.

Base Sequence↗

PubMed2005

Comparison of cryopreservation and standard needle biopsy for gene expression profiling of human breast cancer specimens.

Given the growing importance of molecular profiling of breast cancer, we initiated a small study to test whether human breast biopsies obtained via cryopreservation large core needle biopsy (C-LCNB) provided similar gene expression profiles compared with 'optimally handled' standard large core needle biopsy (S-LCNB) specimens. Five matched pairs of C-LCNB versus S-LCNB were obtained at the same visit, and subjected to gene array expression analysis using the Affymetrix system with U133A chips. No significant changes in gene expression were identified comparing the C-LCNB versus the matched S-LCNB from individual patients. This was corroborated by a paired t-test analysis, which supported the hypothesis that the S/C biopsies measured equivalent samples. A small number of genes (17) showed decreased expression when second biopsies were compared with first biopsies, suggesting a slight patient response to the first biopsy. A scatter plot analysis comparing first biopsy versus second biopsy values disclosed a slope of 0.859, further indicating that the first biopsy affects the second biopsy measurement. It therefore appears that conventional biopsies, when handled appropriately, provide RNA which is equivalent to RNA from biopsies which are frozen immediately, but that multiple biopsy protocols may introduce additional complexities.

Biopsy, Needle↗

PubMed2004

Spontaneous transformation of an immortalized hepatocyte cell line: potential role of a nuclear protease.

In this study, we utilized an in vitro model of spontaneous transformation/progression, an SV40 large T antigen-immortalized rat hepatocyte cell line (designated CWSV14) that is very weakly tumorigenic at low-passage, but acquires a transformed phenotype upon extended passage in cell culture. Here we show that this mid-passage transformation is accompanied by development of aneuploidy and disorganization of the actin cytoskeleton, concomitant with a large increase in a chymotrypsin-like nuclear protease activity which we have previously implicated in chemical transformation of fibroblasts and ras-transformation of hepatocytes. Passage of the CWSV14 cells with AAPF(cmk), a relatively selective inhibitor of the nuclear protease activity, abrogates the acquisition of the transformed phenotype and prevents the changes in the actin cytoskeleton. We hypothesize that the nuclear protease may play a role in initiating development of genomic instability, paralleling the archetypical role of proteases in paradigms such as the SOS-type responses in bacteria and yeast.

Animals↗

PubMed2004

A self-processing ribozyme cassette: utility against human papillomavirus 11 E6/E7 mRNA and hepatitis B virus.

We have been developing a self-processing triple-ribozyme cassette, which consists of two cis-acting hammerhead ribozymes flanking an internal, trans-acting hammerhead ribozyme (ITRz). Here, the single ITRz was replaced by two contiguous ITRz (dITRz), and a short poly(A) tail was designed onto the 3' end of the liberated dITRz, to produce the "SNIP(AA)" cassette. Self-processing of the cassette appeared to proceed efficiently in cells: The only region of the cassette identified in cells was the liberated dITRz, with approximately 10-20% of the dITRz found within the nucleus. We tested this reagent against two therapeutically important targets, human papillomavirus 11 E6/E7 mRNA and hepatitis B virus (HBV). Library selection protocols were utilized to define accessible target sites, and ribozymes targeted to these sites were very active in vitro. Pairs of the selected ribozymes were then inserted into the SNIP(AA) cassette. SNIP(AA) constructs targeted to the E6/E7 mRNA were tested in cell culture using a cotransfection approach. Significant reductions were produced in E6/E7 target, with 80-90% reductions observed at 5 days following cotransfection. SNIP(AA) constructs targeted to HBV RNA were tested in vivo in a transgenic mouse model. SNIP(AA) constructs were packaged in liposomes, which were targeted to hepatocytes using asialofetuin, and administered ip. After 2 weeks, a >80% reduction in viral liver DNA was observed. Immunohistochemical staining for core antigen showed a similar decrease in the number of hepatocytes staining positively, compounded by a concomitant loss of residual staining intensity. These results demonstrate the in vivo utility of the self-processing SNIP(AA) cassette against HBV.

Animals↗

PubMed2004

Identification of efficient cleavage sites in long-target RNAs.

In this chapter, we describe a procedure for identification of efficient hammerhead ribozyme (hRz) cleavage sites in target RNAs. An active hRz library, containing randomized recognition sequences flanked by fixed 5' and 3' regions, is designed to generate enormous diversity. The library is incubated with target RNA at an elevated temperature in the absence of magnesium, and bound library pools are isolated, reamplified, and rebound to target RNA. After two rounds, the active preselected library pool is incubated at 37 degrees C with target RNA in the presence of magnesium, and cleavage products are directly identified on sequencing gels. The protocol identifies highly active hRz, which typically have Kms of 20-80 nM, and kcat/Km values of 10(6).

Base Sequence↗

PubMed2004

Parameters affecting substance P measurement in heart, lung, and skin.

Substance P (SP), a neuropeptide that is widely distributed both peripherally and centrally, mediates several pathophysiological processes. Among current assays for SP, enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) have been most widely used. Several previous studies, mostly performed with nerve extracts or organ perfusates, determined that acidity of the extraction buffer as well as the number extractions performed constitute factors influencing accurate measurements. We used an ELISA protocol in this study to analyze methodological aspects of SP measurement in extracts from heart, skin, and lung. The extraction procedure had two steps, an acid extraction followed by a column extraction. We could effectively measure SP with extract from as little as 10 mg of tissue. For each tissue examined, different variables influenced the SP measured. For all tissues, the weight of tissue extracted was critical; the more tissue extracted, the lower the sensitivity of the assay. This problem could be overcome in skin by omitting the column extraction. When mechanical loses were considered (e.g., loss during extraction and SP retained by the column after elution), column extraction improved SP measurements only with lung tissue. The amount of SP remaining in the sample after the first extraction also varied among tissues. The first acid extraction effectively isolated 80% of total SP from skin. In contrast, the first extraction with lung tissue recovered only 58%. Because both acid and heat effectively release SP from nerve endings, this could reflect the presence of non-neuronal SP, especially in lung. High-dose capsaicin treatment, which depletes SP in nerve endings, caused 42% loss of SP in skin independent of amount of tissue extracted Our results suggest that a second acid extraction of tissue should be performed and that column extraction is clearly detrimental with skin samples.

Animals↗

PubMed2003

Rapid identification of efficient target cleavage sites using a hammerhead ribozyme library in an iterative manner.

A major limitation to the effectiveness of ribozymes is definition of accessible sites in targeted RNAs. Although library selection procedures have been developed, they are generally difficult to perform and have not been widely employed. Here we describe a selection technology that utilizes a randomized, active hammerhead ribozyme (Rz) library in an iterative manner. After two rounds of binding under inactive conditions, the selected, active Rz library is incubated with target RNA, and the sites of cleavage are identified on sequencing gels. We performed this library-selection protocol using human papillomavirus type 16 E6/E7 mRNA as target and constructed Rz targeted to the identified sites. Rz targeted to sites identified with this procedure were generally highly active in vitro and, more importantly, they were highly active in cell culture, whereas their catalytically inactive counterparts were not. This protocol can be used to identify a set of potential target sites within a relatively short time.

Base Sequence↗

PubMed2002

Involvement of the fungal nuclear migration gene nudC human homolog in cell proliferation and mitotic spindle formation.

Essential genes which are required for normal nuclear migration and play a role in developmental processes have been isolated from model genetic organisms. One such gene is nudC (nuclear distribution C), which is required for positioning nuclei in the cytoplasm of the filamentous fungus Aspergillus nidulans and for normal colony growth. This gene is highly conserved, structurally and functionally, throughout evolution and the human homolog, HnudC, has been cloned. To study the function of nudC in higher eukaryotic cells, HnudC was downregulated by developing triple ribozyme constructs, consisting of two cis-acting ribozymes which liberate an internal trans-acting ribozyme targeted to HnudC. Efficient cleavage sites in HnudC mRNA were identified using a library selection technique and HnudC-targeted internal ribozymes were cloned into a triple ribozyme cassette. Triple ribozyme constructs were subcloned into an ecdysone-inducible expression vector and stably transfected into human embryonic 293 cells. Muristerone A induced expression of the HnudC ribozyme and produced specific reduction of HnudC mRNA. Downregulation of HnudC mRNA resulted in significant inhibition of cell proliferation in clones expressing the HnudC-targeted triple ribozyme, which was not observed in uninduced cells or cells transfected with vector alone. In induced cultures, many mitotic cells demonstrated defects in spindle architecture during mitosis. The most common defect observed was multiple mitotic spindle poles rather than the expected bipolar structure. These data demonstrate the fundamental importance of HnudC in eukaryotic cell proliferation and a functional role for HnudC in spindle formation at mitosis.

Cell Cycle Proteins↗

PubMedDate not supplied

Capsaicin-mediated denervation of sensory neurons promotes mammary tumor metastasis to lung and heart.

Capsaicin specifically activates or destroys small diameter nociceptive sensory neurons that contain the capsaicin receptor, also called vanilloid receptor 1. Neurons sensitive to capsaicin mediate inflammatory pain and are important targets for management of chronic pain. These neurons also regulate local tissue homeostasis, inflammation, healing and development, especially under conditions of psychological stress. Stress contributes to increased cancer recurrence and metastasis through as yet undefined mechanisms. Likewise, activity of capsaicin-sensitive neurons is altered by pathological conditions that may lead to metastatic growth (e.g. stress). Therefore, we examined effects of a treatment that induces sensory nerve denervation on breast cancer metastases. Systemic denervation of sensory neurons caused by treatment with 125 mg/kg capsaicin resulted in significantly more lung and cardiac metastases in adult mice injected orthotopically with syngeneic 4T1 mammary carcinoma cells than was observed in vehicle-treated controls. Heart metastases, normally very rare, occurred as pericardial nodules, intra-myocardial nodules, or combined pericardial-myocardial lesions. Since the rate of primary tumor growth was unaffected, effects on metastases appear to be host tissue-specific. Although preliminary, these observations provide one possible explanation for resistance of cardiac tissue to tumor involvement and highlight contributions of host tissue, including sensory neurons, in the efficiency of cancer metastasis.

Animals↗