Biomedical subjects

Christopher J Kemp

Publications and source records attributed to Christopher J Kemp.

16 recordsLinked to original sources

PubMed2026

Ex Vivo Tumor-Derived Organoid Pharmacotyping Identifies Personalized Therapeutic Options for Patients with Biliary Tract Cancer.

UNLABELLED: Biliary tract cancers (BTC) pose clinical challenges due to poor chemotherapy response and aggressive disease course. We evaluated patient-derived tumor organoid-based drug sensitivity testing as a tool to guide therapy. In this multicenter study, 26 tumor organoids were successfully derived from 43 patients with BTC and tested with an average of 50 cancer-directed therapies using the Clinical Laboratory Improvement Amendments-certified PARIS assay. Despite most organoids being from late-stage disease, 24/26 (92.3%) exhibited strong sensitivity to one or more targeted agents. Active drugs included inhibitors of EGFR/HER2, MEK, ERK, BCR-ABL and SRC family, mTOR, PI3K, MDM2, BCL2, and BET. Drug sensitivities aligned with known genetic biomarkers but were also observed in cultures lacking them, indicating ex vivo testing can expand actionability beyond genomics. In five cases, results guided therapy; one patient with an FGFR-BICC1 fusion refractory to FGFR inhibitors responded to dasatinib, achieving symptomatic improvement, stable disease, and >8-month survival. SIGNIFICANCE: Ex vivo drug testing of tumor-derived organoids is clinically feasible and can be used to identify personalized treatment options for patients with BTC, to evaluate the functional relevance of genomic biomarkers, and to guide treatment in real time.

Humans↗

PubMed2006

A pathway in quiescent cells that controls p27Kip1 stability, subcellular localization, and tumor suppression.

We have created two knock-in mouse models to study the mechanisms that regulate p27 in normal cells and cause misregulation of p27 in tumors: p27(S10A), in which Ser10 is mutated to Ala; and p27(CK-), in which point mutations abrogate the ability of p27 to bind cyclins and CDKs. These two mutant alleles identify steps in a pathway that controls the proteasomal degradation of p27 uniquely in quiescent cells: Dephosphorylation of p27 on Ser10 inhibits p27 nuclear export and promotes its assembly into cyclin-CDK complexes, which is, in turn, necessary for p27 turnover. We further show that Ras-dependent lung tumorigenesis is associated with increased phosphorylation on Ser10 and cytoplasmic mislocalization of p27. Indeed, we find that p27(S10A) is refractory to Ras-induced cytoplasmic translocation and that p27(S10A) mice are tumor resistant. Thus, phosphorylation of p27 on Ser10 is an important event in the regulation of the tumor suppressor function of p27.

Amino Acid Substitution↗

PubMed2006

Non-invasive imaging of carcinogen-induced early neoplasia using ultrahigh-resolution optical coherence tomography.

BACKGROUND AND OBJECTIVE: Improved diagnostics capable of non-invasive detection of early stage carcinogenesis would benefit basic research, and potentially aid in clinical cancer diagnosis and management. The two-stage carcinogenesis protocol is widely used for studying the multi-stage nature of tumor development in mice and provides insight into tumor development in other animal models and humans. The objective of this study was to investigate the feasibility of non-invasive optical coherence tomography (OCT) for in vivo imaging of microanatomical changes in the epidermis and dermis during early carcinogenesis using a mouse skin model. MATERIALS AND METHODS: 10 NIH mice were treated with DMBA and TPA following the well-established two-stage carcinogenesis protocol. OCT imaging of treated skin from live mice was performed at five time points (Week 4-8) after tumor initiation to reveal the structural changes in the epidermis and dermis associated with the earliest, premalignant stages of tumor development. OCT images were compared with histology findings. In addition, OCT signals were quantitatively analyzed to evaluate tissue optical property changes during early carcinogenesis. RESULTS: Early structural changes in the epidermis, dermis and hair follicles during carcinogenesis were clearly delineated in vivo using OCT. OCT images correlated well with histological findings. Quantitative OCT signal analysis revealed a statistically significant change in the extinction coefficient for untreated (40.5 +/- 17.0 mm(-1)) and treated (9.6 +/- 3.6 mm(-1)) mouse epidermis (P < 0.005). The dermis extinction coefficient for the treated mouse skin (3.7 +/- 0.9 mm(-1)) was lower than the untreated one (4.7 +/- 1.6 mm(-1)), but was not statistically significant (P > 0.10). Furthermore, the papilloma extinction coefficient (2.9 +/- 0.3 mm(-1)) was significantly lower than the extinction coefficient for the treated epidermis (P < 0.005) and dermis (P < 0.01). CONCLUSION: OCT is a viable tool for assessing the earliest stages of carcinogenesis and has potential for early detection of neoplasia in skin, as well as in epithelial linings of other organs.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2005

Tumor suppressor genetics.

The observation that mutations in tumor suppressor genes can have haploinsufficient, as well as gain of function and dominant negative, phenotypes has caused a reevaluation of the 'two-hit' model of tumor suppressor inactivation. Here we examine the history of haploinsufficiency and tumor suppressors in order to understand the origin of the 'two-hit' dogma. The two-hit model of tumor suppressor gene inactivation was derived from mathematical modeling of cancer incidence. Subsequent interpretations implied that tumor suppressors were recessive, requiring mutations in both alleles. This model has provided a useful conceptual framework for three decades of research on the genetics and biology of tumor suppressor genes. Recently it has become clear that mutations in tumor suppressor genes are not always completely recessive. Haploinsufficiency occurs when one allele is insufficient to confer the full functionality produced from two wild-type alleles. Haploinsufficiency, however, is not an absolute property. It can be partial or complete and can vary depending on tissue type, other epistatic interactions, and environmental factors. In addition to simple quantitative differences (one allele versus two alleles), gene mutations can have qualitative differences, creating gain of function or dominant negative effects that can be difficult to distinguish from dosage-dependence. Like mutations in many other genes, tumor suppressor gene mutations can be haploinsufficient, dominant negative or gain of function in addition to recessive. Thus, under certain circumstances, one hit may be sufficient for inactivation. In addition, the phenotypic penetrance of these mutations can vary depending on the nature of the mutation itself, the genetic background, the tissue type, environmental factors and other variables. Incorporating these new findings into existing models of the clonal evolution will be a challenge for the future.

Animals↗

PubMed2005

A software suite for the generation and comparison of peptide arrays from sets of data collected by liquid chromatography-mass spectrometry.

There is an increasing interest in the quantitative proteomic measurement of the protein contents of substantially similar biological samples, e.g. for the analysis of cellular response to perturbations over time or for the discovery of protein biomarkers from clinical samples. Technical limitations of current proteomic platforms such as limited reproducibility and low throughput make this a challenging task. A new LC-MS-based platform is able to generate complex peptide patterns from the analysis of proteolyzed protein samples at high throughput and represents a promising approach for quantitative proteomics. A crucial component of the LC-MS approach is the accurate evaluation of the abundance of detected peptides over many samples and the identification of peptide features that can stratify samples with respect to their genetic, physiological, or environmental origins. We present here a new software suite, SpecArray, that generates a peptide versus sample array from a set of LC-MS data. A peptide array stores the relative abundance of thousands of peptide features in many samples and is in a format identical to that of a gene expression microarray. A peptide array can be subjected to an unsupervised clustering analysis to stratify samples or to a discriminant analysis to identify discriminatory peptide features. We applied the SpecArray to analyze two sets of LC-MS data: one was from four repeat LC-MS analyses of the same glycopeptide sample, and another was from LC-MS analysis of serum samples of five male and five female mice. We demonstrate through these two study cases that the SpecArray software suite can serve as an effective software platform in the LC-MS approach for quantitative proteomics.

Amino Acid Sequence↗

PubMed2005

Deficiency in the gap junction protein connexin32 alters p27Kip1 tumor suppression and MAPK activation in a tissue-specific manner.

Connexin32 knockout mice (Cx32-KO) exhibit increased chemical- and radiation-induced liver and lung tumor formation with many lung tumors demonstrating decreased levels of the tumor suppressor p27KIP1. To determine if p27 deficiency alters Cx32-influenced tumorigenesis, we have generated a Cx32/p27 double-deficient mouse strain (DKO) and show here that exposure of these mice to X-ray radiation resulted in an increase or decrease in tumorigenesis depending on the tissue. Several tissues were highly sensitive to loss of p27 tumor suppressor function (intestine, adrenal, pituitary) resulting in an increased overall tumor burden in DKO mice compared to both wild-type (P<0.005) and Cx32-KO mice (P=0.066). However, additional deletion of p27 in a Cx32-KO background resulted in a statistically significant decrease in the liver tumor incidence suggesting that Cx32 and p27 pathways mechanistically interact. Immunohistochemical analysis revealed an increased percentage of Cx32-KO liver and lung tumors harboring active mitogen-activated protein kinase (Erk1, Erk2) pathways in contrast to lower percentages of activated wild-type (P<0.005) and DKO tumors (P=0.027). Increased MAPK activation in liver tumors did not correlate with Ha-ras codon-61 mutation status. This study demonstrates that tissues dependent on Cx32 tumor suppression, such as the liver and lung, exhibit altered tumorigenesis and tumor biology (MAPK pathway activation) related to p27 status.

Adenoma↗

PubMed2005

Multistep skin cancer in mice as a model to study the evolution of cancer cells.

Although much of cancer research relies on Nowell's clonal evolution hypothesis as a conceptual framework, large gaps remain in understanding how tumors develop. The multistage skin cancer model in mice provides continuing insight on fundamental aspects of tumor evolution. In this model, mutation of the oncogene Hras is frequently the initiating event while mutation of the tumor suppressor p53 is a late event, associated with malignant progression. Recent evidence demonstrates that intracellular signaling from the initial Hras mutation leads directly to the activation of p53, creating selective pressure in favor of cells with mutant p53. Thus, selection for subsequent mutations is mechanistically linked to the initial mutation, explaining the preferred order of mutational events observed. Analysis of this model also reveals that a diverse array of signals can selectively impair or enhance clonal expansion of Ras mutant cells into a visible neoplasm. These modifiers can be genetic, physiological, or environmental and are often highly specific to tumor cells. This indicates that tumor cells have an inherent reduced capacity to buffer against perturbations. Reduced buffering may play an important role in both tumor evolution and therapy response and may be a hallmark of cancer cells.

Animals↗

PubMed2004

High throughput quantitative analysis of serum proteins using glycopeptide capture and liquid chromatography mass spectrometry.

It is expected that the composition of the serum proteome can provide valuable information about the state of the human body in health and disease and that this information can be extracted via quantitative proteomic measurements. Suitable proteomic techniques need to be sensitive, reproducible, and robust to detect potential biomarkers below the level of highly expressed proteins, generate data sets that are comparable between experiments and laboratories, and have high throughput to support statistical studies. Here we report a method for high throughput quantitative analysis of serum proteins. It consists of the selective isolation of peptides that are N-linked glycosylated in the intact protein, the analysis of these now deglycosylated peptides by liquid chromatography electrospray ionization mass spectrometry, and the comparative analysis of the resulting patterns. By focusing selectively on a few formerly N-linked glycopeptides per serum protein, the complexity of the analyte sample is significantly reduced and the sensitivity and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptides from unfractionated samples. We provide data that document the performance of the method and show that sera from untreated normal mice and genetically identical mice with carcinogen-induced skin cancer can be unambiguously discriminated using unsupervised clustering of the resulting peptide patterns. We further identify, by tandem mass spectrometry, some of the peptides that were consistently elevated in cancer mice compared with their control littermates.

Animals↗

PubMed2004

p19Arf suppresses growth, progression, and metastasis of Hras-driven carcinomas through p53-dependent and -independent pathways.

Ectopic expression of oncogenes such as Ras induces expression of p19(Arf), which, in turn, activates p53 and growth arrest. Here, we used a multistage model of squamous cell carcinoma development to investigate the functional interactions between Ras, p19(Arf), and p53 during tumor progression in the mouse. Skin tumors were induced in wild-type, p19(Arf)-deficient, and p53-deficient mice using the DMBA/TPA two-step protocol. Activating mutations in Hras were detected in all papillomas and carcinomas examined, regardless of genotype. Relative to wild-type mice, the growth rate of papillomas was greater in p19(Arf)-deficient mice, and reduced in p53-deficient mice. Malignant conversion of papillomas to squamous cell carcinomas, as well as metastasis to lymph nodes and lungs, was markedly accelerated in both p19 (Arf)- and p53-deficient mice. Thus, p19(Arf) inhibits the growth rate of tumors in a p53-independent manner. Through its regulation of p53, p19(Arf) also suppresses malignant conversion and metastasis. p53 expression was upregulated in papillomas from wild-type but not p19( Arf)-null mice, and p53 mutations were more frequently seen in wild-type than in p19( Arf)-null carcinomas. This indicates that selection for p53 mutations is a direct result of signaling from the initiating oncogenic lesion, Hras, acting through p19(Arf).

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2004

Distinct roles for p53, p27Kip1, and p21Cip1 during tumor development.

Mutations in p53 and reduced expression of the Cdk inhibitor p27Kip1 are frequently observed in a wide variety of human cancers, but it is not known whether alterations in these tumor suppressors interact to influence tumor progression. To address this, we measured tumor latency and spectrum in p53 and p27 single and compound mutant mice. p53-/- (null) mice developed T-cell lymphomas and soft-tissue sarcomas, while p27-/- mice developed adenomas of the pituitary and lung, but with much longer latency. The latency for tumor development in p53-/- p27-/- and p53-/- p27+/- compound mutant mice was significantly reduced, by 15-30%, compared to single mutant p53-/- mice. The tumor spectrum in the compound mutants was similar to that of p53-/- mice, and additional tumors of diverse histotypes. In tumors from p53-/- mice, p27 protein levels were reduced to a greater extent than were mRNA levels, indicating that p27 is downregulated in tumors at the transcriptional as well as post-transcriptional levels. In contrast, mice deficient in another Cdk inhibitor p21Cip1, which is also a transcriptional target and effector of p53, showed only a marginal increase in tumor predisposition in response to ENU treatment. Thus, downregulation of p27 is a common feature in p53-/- tumors. Germline deletion of one or both alleles of p27 accelerates tumor development and associated mortality in p53-/- mice, indicating potent synergy between loss of p27 and p53. Although p21 is functionally similar to both p53 and p27, it plays a lesser role in tumor suppression. These results further highlight the highly cooperative nature of p27 and its central role in tumor suppression.

Animals↗

PubMed2002

Selective inhibition of the epidermal growth factor receptor impairs intestinal adaptation after small bowel resection.

BACKGROUND: Prior indirect studies have suggested that a functional epidermal growth factor receptor (EGFR) appears to be indispensable for the adaptive response of the remnant intestine to massive small bowel resection (SBR). The recent availability of a specific pharmacologic EGFR inhibitor enabled us to more directly test the hypothesis that EGFR signaling is required for postresection intestinal adaptation. METHODS: Mice (C57B1/6, n = 26) underwent a 50% SBR or sham operation and were then given orogastric EGFR inhibitor (ZD1839, 50 mg/kg/day) or vehicle. After 3 days, indices of adaptation (wet weight, crypt depth, and villus height) and apoptotic index (number of apoptotic bodies per crypt) were calculated in the ileum. The expression of proliferating cell nuclear antigen (PCNA) and activated EGFR was measured by Western blotting. RESULTS: ZD1839 prevented EGFR activation and the normal postresection increases in ileal wet weight, villus height, and crypt depth. Enterocyte proliferation was reduced twofold in the SBR group by ZD1839. Although not statistically significant, rates of enterocyte apoptosis were the highest in the inhibitor-treated mice. CONCLUSION: Following massive SBR, pharmacologic inhibition of the EGFR attenuates proliferation and the normal adaptive response of the intestine. These results more directly confirm the requirement of a functional EGFR as a mediator of the postresection adaptation response. This study demonstrates an in vivo application of a novel selective EGFR inhibitor and offers a unique experimental model to gain mechanistic insight into understanding postresection intestinal adaptation.

Adaptation, Physiological↗

PubMed2002

Intestinal and hepatic response to combined partial hepatectomy and small bowel resection in mice.

BACKGROUND: Both partial-hepatectomy (PHx) and massive small bowel resection (SBR) are strong mitogenic signals to the remnant liver and intestine, respectively. This study tested the hypothesis that PHx was an additive signal for intestinal adaptation after massive SBR. METHODS: Male mice underwent either sham SBR or 50% proximal SBR. Mice from these two groups were then subjected to a 70% PHx or sham PHx. After 3 days, parameters of intestinal adaptation and liver regeneration were recorded in the remnant intestine and liver, respectively. RESULTS: Intestinal adaptation following SBR occurred normally, but was not enhanced after concomitant PHx. On the other hand, SBR impaired the regenerative ability of the liver following PHx. CONCLUSIONS: Intestinal adaptation after SBR takes priority over liver regeneration after PHx. These data implicate a hierarchy with regard to adaptive alterations to organ loss and endorse an important role for the intestinal mucosa in the regulation of hepatic regeneration.

Adaptation, Physiological↗

PubMed2002

Pathway-specific tumor suppression. Reduction of p27 accelerates gastrointestinal tumorigenesis in Apc mutant mice, but not in Smad3 mutant mice.

Expression of the cyclin-dependent kinase inhibitor p27(Kip1) (p27) is frequently reduced in human colorectal cancer, and this correlates with poor patient prognosis. To clarify the role of p27 in gastrointestinal (GI) cancer, we measured p27 expression, as well as the effect of germline deletion of p27, in 3 different mouse models of GI neoplasia. p27 expression was frequently reduced in GI tumors arising in 1,2-dimethylhydrazine (DMH) treated mice, and in Apc mutant Min/+ mice, but not in GI tumors arising in Smad3 mutant mice. Germline deletion of p27 resulted in accelerated tumor development and increased tumor cell proliferation in both DMH treated and Min/+ mice, but not in Smad3 mutant mice. p27 deficiency also led to increased adenoma to adenocarcinoma progression. These results indicate that reduction of p27 cooperates with mutations in Apc but not in Smad3 during GI tumorigenesis. Thus, tumor suppression by p27 is contingent on the specific oncogenic pathway that drives tumor development.

1,2-Dimethylhydrazine↗

PubMed2002

Intestinal permeability and bacterial translocation are uncoupled after small bowel resection.

BACKGROUND/PURPOSE: Gut barrier failure and bacterial translocation have been proposed to cause infection and sepsis in patients with the short bowel syndrome. This study tested the hypothesis that permeability is increased in the adapting remnant ileum after massive small bowel resection (SBR). METHODS: Male ICR mice underwent a 50% proximal SBR or sham operation. At 3, 7, and 14 days, the ileum was mounted in an Ussing chamber. Mucosal-to-serosal flux of low (dextran) and high (horseradish peroxidase; HRP) molecular weight markers was determined. Additionally, bacterial translocation was measured by culturing blood, mesenteric lymph nodes, liver, and spleen at 3 and 14 days after SBR or sham operation. RESULTS: Permeability to dextran was reduced immediately after SBR but was no different at later time-points. HRP permeability was no different at any time-point. Translocation of Gram-negative bacteria to the mesenteric lymph nodes and liver was more frequent in the SBR animals 3 and 14 days postoperatively. CONCLUSIONS: Intestinal permeability to macromolecules is not increased after massive SBR, but the rate of translocation to the mesenteric lymph nodes and liver is elevated. This suggests that the mechanism for bacterial translocation after SBR does not involve alterations in gut permeability.

Adaptation, Biological↗

PubMed2002

Role of epidermal growth factor and its receptor in chemotherapy-induced intestinal injury.

Several growth factors are trophic for the gastrointestinal tract and able to reduce the degree of intestinal damage caused by cytotoxic agents. However, studies of epidermal growth factor (EGF) for chemotherapy-induced intestinal injury are conflicting. The development of a transgenic mouse that specifically overexpresses EGF in the small intestine provided a unique opportunity to assess the contribution of EGF in mucositis. After a course of fluorouracil, transgenic mice fared no better than control mice. Weight recovery was inferior, and mucosal architecture was not preserved. Apoptosis was not decreased and proliferation was not increased in the crypts. To corroborate the findings in transgenic mice, ICR mice were treated with exogenous EGF after receiving fluorouracil. Despite ileal upregulation of native and activated EGF receptor, the mice were not protected from intestinal damage. No benefits were observed with different EGF doses or schedules or routes of EGF administration. Finally, mucositis was induced in mutant mice with specific defects of the EGF signaling axis. Compared with control mice, clinical and histological parameters of intestinal injury after fluorouracil were no different in waved-2 mice, which have functionally diminished EGF receptors, or waved-1 mice, which lack transforming growth factor-alpha, another major ligand for the EGF receptor. These findings do not support a critical role for EGF or its receptor in chemotherapy-induced intestinal injury.

Animals↗

PubMedDate not supplied

p27(Kip1) (Cdkn1b)-deficient mice are susceptible to chemical carcinogenesis and may be a useful model for carcinogen screening.

The two-year rodent bioassay is one of several tests that are widely used by governmental regulatory agencies as well as pharmaceutical and chemical companies to determine the carcinogenic potential of chemicals or environmental agents where human exposure is anticipated. That this assay has remained relatively unchanged for the last 25 years is a testament to the power of this approach to identify carcinogens and thus to minimize human exposure. However, there has long been controversy over the specificity and relevance of the rodent bioassay as well as its high cost in terms of time, expense, and numbers of mice. Much discussion has been generated in recent years over how to improve the 2-year rodent bioassay for more accurate and faster detection of potential human carcinogens. Here, we argue that the use of p27(Kip1) (Cdkn1b) knockout mouse for carcinogen screening may solve several shortcomings inherent in the conventional bioassay while preserving its best quality, that is, protecting public health by providing reliable in vivo information on the potential of chemicals to cause cancer.

Animals↗