The effect of Nullomer-derived peptides 9R, 9S1R and 124R on the NCI-60 panel and normal cell lines.

BACKGROUND: Nullomer peptides are the smallest sequences absent from databases of natural proteins. We first began compiling a list of absent 5-amino acid strings in 2006 (1). We report here the effects of Nullomer-derived peptides 9R, 9S1R and 124R on the NCI-60 panel, derived from human cancers of 9 organs (kidney, ovary, skin melanoma, lung, brain, lung, colon, prostate and the hematopoietic system), and four normal cell lines (endothelial HUVEC, skin fibroblasts BJ, colon epithelial FHC and normal prostate RWPE-1). METHODS: NCI-60 cancer cell panel and four normal cell lines were cultured in vitro in RPMI1640 supplemented with 10% Hyclone fetal bovine serum and exposed for 48 h to 5 µM, 25 µM and 50 µM of peptides 9R, 9S1R and 124R. Viability was assessed by CCK-8 assay. For peptide ATP depletion effects, one cell line representing each organ in the NCI-60 panel, and four normal cell lines were exposed to 50 µM of peptides 9R, 9S1R and 124R for 3 h. The ATP content was assessed in whole cells, and their supernatants. RESULTS: Peptides 9S1R and 9R are respectively lethal to 95 and 81.6% of the 60 cancer cell lines tested. Control peptide 124R has no effect on the growth of these cells. Especially interesting the fact that peptides 9R and 9S1R are capable of killing drug-resistant and hormone-resistant cell lines, and even cancer stem cells. Peptides 9R and 9S1R have a broader activity spectrum than many cancer drugs in current use, can completely deplete cellular ATP within 3 h, and are less toxic to 3 of the 4 normal cell lines tested than they are to several cancers. CONCLUSIONS: Nullomer peptides 9R and 9S1R have a large broad lethal effect on cancer cell lines derived from nine organs represented in the NCI-60 panel. This broad activity crosses many of the categorical divisions used in the general classification of cancers: solid vs liquid cancers, drug sensitive vs drug resistant, hormone sensitive vs hormone resistant, cytokine sensitive vs cytokine non sensitive, slow growing vs rapid growing, differentiated vs dedifferentiated cancers. Furthermore peptides 9R and 9S1R are lethal to cancer stem cells and breast canrcinosarcoma.

Safeguarding forensic DNA reference samples with nullomer barcodes.

Unintended transfer of biological material containing DNA is a concern to all laboratories conducting PCR analysis. While forensic laboratories have protocols in place to reduce the possibility of contaminating casework samples, there is no way to detect when a reference sample is mislabeled as evidence, or contaminates a forensic sample. Thus there is public concern regarding the safeguarding of DNA submitted to crime labs. We demonstrate a method of introducing an internal amplification control to reference samples, in the form of a nullomer barcode which is based upon sequences absent or rare from publically accessible DNA databases. The detection of this barcode would indicate that the source of analyzed DNA was from a reference sample provided by an individual, and not from an evidence sample. We demonstrate that the nullomers can be added directly to collection devices (FTA paper) to allow tagging during the process of sample collection. We show that such nullomer oligonucleotides can be added to existing forensic typing and quantification kits, without affecting genotyping or quantification results. Finally, we show that even when diluted a million-fold and spilled on a knife, the nullomer tags can be clearly detected. These tags support the National Research Council of the National Academy recommendation that "Quality control procedures should be designed to identify mistakes, fraud, and bias" in forensic science (National Academy of Sciences, 2009).

Nullomer derived anticancer peptides (NulloPs): differential lethal effects on normal and cancer cells in vitro.

We demonstrate the first use of the nullomer (absent sequences) approach to drug discovery and development. Nullomers are the shortest absent sequences determined in a species, or group of species. By identifying the shortest absent peptide sequences from the NCBI databases, we screened several potential anti-cancer peptides. In order to improve cell penetration and solubility we added short poly arginine tails (5Rs), and initially solubilized the peptides in 1M trehalose. The results for one of the absent sequences 9R (RRRRRNWMWC), and its scrambled version 9S1R (RRRRRWCMNW) are reported here. We refer to these peptides derived from nullomers as PolyArgNulloPs. A control PolyArgNulloP, 124R (RRRRRWFMHW), was also included. The lethal effects of 9R and 9S1R are mediated by mitochondrial impairment as demonstrated by increased ROS production, ATP depletion, cell growth inhibition, and ultimately cell death. These effects increase over time for cancer cells with a concomitant drop in IC-50 for breast and prostate cancer cells. This is in sharp contrast to the effects in normal cells, which show a decreased sensitivity to the NulloPs over time.

Microfluidic devices integrating microcavity surface-plasmon-resonance sensors: glucose oxidase binding-activity detection.

We have developed miniature (approximately 1 microm diameter) microcavity surface-plasmon-resonance sensors (MSPRS), integrated them with microfluidics, and tested their sensitivity to refractive-index changes. We tested their biosensing capability by distinguishing the interaction of glucose oxidase (M(r) 160 kDa) with its natural substrate (beta-D-glucose, M(r) 180 Da) from its interactions with nonspecific substrates (L-glucose, D-mannose, and 2-deoxy-D-glucose). We ran the identical protocol we had used with the MSPRS on a Biacore 3000 instrument using their bare gold chip. Only the MSPRS was able to detect beta-D-glucose binding to glucose oxidase. Each MSPRS can detect the binding to its surface of fewer than 35,000 glucose oxidase molecules (representing 9.6 fg or 60 zmol of protein), about 10(6) times fewer than classical surface-plasmon-resonance biosensors.

Mitochondrial impairment is a critical event in anthrax lethal toxin-induced cytolysis of murine macrophages.

Numerous early events in anthrax lethal toxin (LT)-mediated cell killing have been described, including uptake of LT and MAPKK cleavage. However, critical downstream events in LT killing remain to be identified. In this study we present evidence that LT causes mitochondrial dysfunction in murine J774A.1 macrophages, as indicated by a continuous drop in both mitochondrial membrane potential and SDH activity. This was further supported by ultrastructural analysis revealing LT-induced swelling of mitochondria. Mitochondrial impairment and cytolysis were controlled by proteasomes in LT-treated macrophages: proteasome inhibitors restored mitochondrial activity and rescued cells from cytolysis, even when added immediately prior to membrane perturbation. Similar to proteasome inhibitors, KCl also efficiently blocked LT-mediated cytolysis, even after late addition. However, KCl did not prevent mitochondrial impairment, though it precluded events linked to LT-induced cytolysis. These events included a precipitous drop in ATP levels and ubiquitinated proteins, revealing that they are epiphenomena in LT killing. Our studies suggest that proteasomes and potassium control LT-induced mitochondrial dysfunction and membrane perturbation, key events in LT killing.

Anthrax lethal toxin-mediated killing of human and murine dendritic cells impairs the adaptive immune response.

Many pathogens have acquired strategies to combat the immune response. Bacillus anthracis interferes with host defenses by releasing anthrax lethal toxin (LT), which inactivates mitogen-activated protein kinase pathways, rendering dendritic cells (DCs) and T lymphocytes nonresponsive to immune stimulation. However, these cell types are considered resistant to killing by LT. Here we show that LT kills primary human DCs in vitro, and murine DCs in vitro and in vivo. Kinetics of LT-mediated killing of murine DCs, as well as cell death pathways induced, were dependent upon genetic background: LT triggered rapid necrosis in BALB/c-derived DCs, and slow apoptosis in C57BL/6-derived DCs. This is consistent with rapid and slow killing of LT-injected BALB/c and C57BL/6 mice, respectively. We present evidence that anthrax LT impairs adaptive immunity by specifically targeting DCs. This may represent an immune-evasion strategy of the bacterium, and contribute to anthrax disease progression. We also established that genetic background determines whether apoptosis or necrosis is induced by LT. Finally, killing of C57BL/6-derived DCs by LT mirrors that of human DCs, suggesting that C57BL/6 DCs represent a better model system for human anthrax than the prototypical BALB/c macrophages.

ATR/TEM8 is highly expressed in epithelial cells lining Bacillus anthracis' three sites of entry: implications for the pathogenesis of anthrax infection.

Anthrax is a disease caused by infection with spores from the bacteria Bacillus anthracis. These spores enter the body, where they germinate into bacteria and secrete a tripartite toxin that causes local edema and, in systemic infections, death. Recent studies identified the cellular receptor for anthrax toxin (ATR), a type I membrane protein. ATR is one of the splice variants of the tumor endothelial marker 8 (TEM8) gene. ATR and TEM8 are identical throughout their extracellular and transmembrane sequence, and both proteins function as receptors for the toxin. ATR/TEM8 function and expression have been associated with development of the vascular system and with tumor angiogenesis. TEM8 is selectively upregulated in endothelial cells during blood vessel formation and tumorigenesis. However, selective expression of TEM8 in endothelial cells contradicts the presumably ubiquitous expression of the receptor. To resolve this controversial issue, we evaluated the distribution of ATR/TEM8 in a variety of tissues. For this purpose, we generated and characterized a novel anti-ATR/TEM8 polyclonal antibody. Here, we show that this novel antibody recognizes all three ATR/TEM8 isoforms, which are widely and differentially expressed in various tissue types. We found that ATR/TEM8 expression is not only associated with tumor endothelial cells, as previously described. Indeed, ATR/TEM8 is highly and selectively expressed in the epithelial cells lining those organs that constitute the anthrax toxin's sites of entry, i.e., the lung, the skin, and the intestine. In fact, we show that ATR/TEM8 is highly expressed in the respiratory epithelium of the bronchi of the lung and is particularly abundant in the ciliated epithelial cells coating the bronchi. Furthermore, immunostaining of skin biopsies revealed that ATR/TEM8 is highly expressed in the keratinocytes of the epidermis. Finally, we show that the epithelial cells lining the small intestine strongly express ATR/TEM8 isoforms. This is the first demonstration that the ATR/TEM8 protein is highly expressed in epithelial cells, which represent the primary location for bacterial invasion. These results suggest that the ATR/TEM8 expression pattern that we describe here is highly relevant for understanding the pathogenesis of anthrax infection.

Interleukin-2 and cancer: critical analysis of results, problems and expectations.

The cancer process in a combination of two kinds of events: a multistep cellular genetic defects giving cells independent growth and great adaptation capability, a multistep interactions profiles with what is called the stromal reaction from the original in situ tumor to the invasive metastatic and angiogenic tumor. The immune system plays an important role in the control of the cancer process but always must be seen as a part integrated in the stromal reaction. In order to boost the immune system capability to treat a cancer we must never forget these cellular and tissular dimensions. Interleukins, growth factors and monoclonal antibodies are new agents are able to bring immunotherapy of cancer to reality. Interleukin 2 did not match our dreams of the ideal factor which can stimulate the defective immune system and bring the cancer evolution to an end. The little but real remissions obtained with the IL-2 high dose protocols still sustains our trust of the immune system as a critical barrier to cancer evolution but the numerous side effects reminds us that cytokines are not to be used as antibiotics and hormones. IL-2 is a regulator of the immune system at the microenvironment level, therefore flooding the blood circulation with high IL-2 doses is not appropriate. We have also to understand that IL-2 can interact directly with cancer cells and also with stromal cells (endothelial and fibroblastic cells), the outcome of IL-2 immunotherapy is not restricted to the interactions with immune cells.