Temozolomide- and fotemustine-induced apoptosis in human malignant melanoma cells: response related to MGMT, MMR, DSBs, and p53.

Malignant melanomas are highly resistant to chemotherapy. First-line chemotherapeutics used in melanoma therapy are the methylating agents dacarbazine (DTIC) and temozolomide (TMZ) and the chloroethylating agents BCNU and fotemustine. Here, we determined the mode of cell death in 11 melanoma cell lines upon exposure to TMZ and fotemustine. We show for the first time that TMZ induces apoptosis in melanoma cells, using therapeutic doses. For both TMZ and fotemustine apoptosis is the dominant mode of cell death. The contribution of necrosis to total cell death varied between 10 and 40%. The O(6)-methylguanine-DNA methyltransferase (MGMT) activity in the cell lines was between 0 and 1100 fmol mg(-1) protein, and there was a correlation between MGMT activity and the level of resistance to TMZ and fotemustine. MGMT inactivation by O(6)-benzylguanine sensitized all melanoma cell lines expressing MGMT to TMZ and fotemustine-induced apoptosis, and MGMT transfection attenuated the apoptotic response. This supports that O(6)-alkylguanines are critical lesions involved in the initiation of programmed melanoma cell death. One of the cell lines (MZ7), derived from a patient subjected to DTIC therapy, exhibited a high level of resistance to TMZ without expressing MGMT. This was related to an impaired expression of MSH2 and MSH6. The cells were not cross-resistant to fotemustine. Although these data indicate that methylating drug resistance of melanoma cells can be acquired by down-regulation of mismatch repair, a correlation between MSH2 and MSH6 expression in the different lines and TMZ sensitivity was not found. Apoptosis in melanoma cells induced by TMZ and fotemustine was accompanied by double-strand break (DSB) formation (as determined by H2AX phosphorylation) and caspase-3 and -7 activation as well as PARP cleavage. For TMZ, DSBs correlated significantly with the apoptotic response, whereas for fotemustine a correlation was not found. Melanoma lines expressing p53 wild-type were more resistant to TMZ and fotemustine than p53 mutant melanoma lines, which is in marked contrast to previous data reported for glioma cells treated with TMZ. Overall, the findings are in line with the model that in melanoma cells TMZ-induced O(6)-methylguanine triggers the apoptotic (and necrotic) pathway through DSBs, whereas for chloroethylating agents apoptosis is triggered in a more complex manner.

Small-molecule Bcl-2 inhibitors sensitise tumour cells to immune-mediated destruction.

The cytotoxic effects of anticancer immune cells are mediated by perforin/granzyme-B, Fas ligand and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), and therefore depend on intact apoptotic responses in target tumour cells. As killing by all three of these mechanisms is blocked by the frequently overexpressed antiapoptotic oncoprotein Bcl-2, we hypothesised that coexposure to a Bcl-2 inhibitor might enhance anticancer immune responses. We evaluated this in U937 lymphoma cells, and A02 melanoma cells, which both show strong Bcl-2 expression. Valpha24(+) Vbeta11(+) natural killer T (NKT) cells expanded from peripheral blood of normal donors (n=3) were coincubated with PKH26-labelled U937 cells, and cytotoxicity was determined by flow cytometry after annexin-V-FITC and 7-AAD staining. In all cases, addition of the HA14-1 small-molecule Bcl-2 inhibitor to the cocultures significantly increased apoptosis in the target U937 cells. Using a similar assay, killing of A02 cells by the cytotoxic T-lymphocyte clone 1H3 was shown to be amplified by coexposure to the potent small-molecule Bcl-2 inhibitor ABT-737. Experiments with immune effectors preincubated with concanamycin-A suggested that sensitisation to perforin/granzyme-B may underlie enhanced target-cell killing observed in the presence of Bcl-2 inhibitors. We conclude that immune destruction of malignant cells can be amplified by molecular interventions that overcome Bcl-2-mediated resistance to apoptosis.

Cross-presentation of a human malaria CTL epitope is conformation dependent.

Little is known about the role of conformation on the antigen processing by antigen presenting cells. Using a well-defined antigen containing two disulfide bridges, the synthetic C-terminal fragment 282-383 derived from Plasmodium falciparum circumsporozoite protein (PfCS 282-383), we show that the reduced form is presented in vitro more efficiently than its oxidized counterpart, inducing stronger CTL recognition. In addition, only the reduced form can be presented by the TAP independent T2 cell line. Thus, the reduced form is processed by TAP dependent and independent pathways.

Treatment for severe palatoclonus by occlusion of the eustachian tube.

PURPOSE: Surgical blocking of the eustachian tube is presented as an ultimate treatment option in a 11-year-old suicidal boy with a therapy-resistant, persistent clicking tinnitus caused by myoclonus of the levator veli palatini. PATIENT: An 11-year-old boy decompensated psychologically as a result of loud and objective tinnitus. The tinnitus could be heard easily by an examiner by bringing his own ear at a distance of approximately 20 to 30 cm to the left ear of the patient. No neurologic etiology for the tinnitus could be traced. Pediatric psychiatric evaluation resulted in a recommendation to perform, as a last resort, an experimental surgical option like blockage of the eustachian tube. INTERVENTION: Treatment with Tegretol (Novartis, The Netherlands) had no effect. Treatment with Dysport (Ipsen) botulin toxin with 30 to 60 U was temporarily effective. Finally, 60 U were not effective anymore. As last refugium, a surgical blockage of the eustachian tube has been performed, first with bone cement and later by a more conventional surgical blockage of that bony tube. OUTCOME: After surgical blockage of the bony part of the eustachian tube, the objective tinnitus disappeared. Blockage of the protympanum by bone cement resulted in only 1 year of successful blocking. After recurrence of the tinnitus combined with aeration of the middle ear, a second surgical transcanal approach was successful in blocking the eustachian tube. With a grommet, the hearing level remained within 10 dB for 0.5 to 8.0 kHz.

Serving up food safety: who wants a piece of the pie?

A total of 12 federal agencies, plus their state counterparts, contribute to the regulatory snarl that governs the safety of the American food supply. With so much federal oversight, one might expect U.S. foods to be virtually risk-free. But this is hardly the case; contaminated food is responsible for 75 million illnesses, 325,000 hospitalizations, and 5,000 deaths in the United States each year. Recent reports from the General Accounting Office and the National Research Council claim that creation of a single agency with centralized authority is the best solution to U.S. food safety problems. Some experts agree that regulatory gaps in food safety highlight the need for centralized leadership, and that more money is necessary to fund the number of inspectors needed to adequately inspect the food supply before it reaches consumers. The single-agency concept has garnered congressional, industry, and scientific support, but the idea isn't without its skeptics, who believe that consolidating food safety under a single agency eliminates checks and balances offered by the current system and, more importantly, runs the risk of politicizing the agency.

Green trees for greenhouse gases: a fair trade-off?

While forests retain carbon in plants, detritus, and soils, utility companies spew it into the air as carbon dioxide, the main greenhouse gas behind global warming. Industrial carbon dioxide emissions aren't currently regulated by federal law, but a number of companies are trying to address the problem voluntarily by launching carbon sequestration programs in heavily forested countries, where carbon is contained in so-called sinks. But the November 2000 meeting of the Kyoto Protocol delegates in The Hague collapsed over the issue of the acceptability of carbon sinks as a source of carbon pollution credits, delivering what many see as a deathblow to the concept. At issue are a host of ecological and statistical questions, differing local land use practices, cultural factors, issues of verifiability, and even disagreement over definitions of basic terms such as "forest" Kyoto negotiators are gearing up for another round of discussions in Bonn in May 2001, and it is likely that the continuing debate over carbon sinks will dominate the agenda.

Charting the map of life.

Scientists expect that mapping the human genome will lead to a host of innovations in biology and research. For example, it may become possible to use DNA microarrays to accurately diagnose cancer and infectious disease subtypes and to predict clinical outcomes. Scientists might also use the genome to look at the interactions of the environment, genetic makeup, and toxic exposures, including the ability of certain beneficial genes to detoxify the body and resist disease. But despite the great potential of the field of genomics, scientists caution that public expectations need to be tempered by reality. People are as much a product of their environment as they are of their genes, say experts, and to suggest that genetics is the sole determinant that defines humans as individuals stretches the science beyond the current data.

Personality predictors of injury-related posttraumatic stress disorder.

This longitudinal, cohort study examined the effect of personality traits on the emergence of posttraumatic stress disorder (PTSD) in a recently traumatized, civilian, mixed-gender sample with significant injuries. Burn survivors (N = 70) were administered the NEO-Personality Inventory (NEO-PI) and the Structured Clinical Interview for DSM III-R (SCID) at hospital discharge and readministered the SCID 4 and 12 months later. Overall, the sample of burn survivors scored significantly higher on neuroticism and extraversion and lower on openness, agreeableness, and conscientiousness relative to a normative national sample. Furthermore, multivariate analysis of variance revealed that PTSD symptom severity groups (i.e., single symptom, multiple symptoms, subthreshold PTSD, PTSD) were differentially related to neuroticism and extraversion. Planned comparisons indicated that neuroticism was higher and extraversion was lower in those who developed PTSD compared with those who did not develop PTSD.

Bordering on environmental disaster.

Millions of people live on the U.S.-Mexico border, drawn by employment opportunities from industry and agriculture, and booming growth is expected to continue in the coming decades. But the influx of people has long outstripped the capacity of border towns and cities to provide for their citizens, and a large percentage of the population lives in sprawling, underserviced shantytowns. Although investment in environmental infrastructure on both sides has led to improvements, shortages among necessities such as drinking water, wastewater treatment, and solid waste disposal are at crisis levels. In addition, a host of diseases including gastrointestinal infections, asthma, tuberculosis, multiple myeloma, systemic lupus erythematosus, hepatitis A, neural tube defects, and lead poisoning have been linked to environmental conditions on the border. Ongoing binational studies are attempting to define the breadth of the public and environmental health crisis and find ways to help make the border a healthier place.

Obtaining fingerprints from mummified fingers: a method for tissue rehydration adapted from the archeological literature.

Our laboratory was asked to help with the rehydration of mummified human fingertips that had been removed from a recently deceased, unidentified female. Using a solution that was found in the archeological literature, we were able to successfully rehydrate dermal tissues to the extent that fingerprints could be taken. We believe that this solution, which until now has not been described in the forensic literature, is effective, affordable, and relatively easy to produce and use.

Lessons from the flood: will Floyd change livestock farming?

When Hurricane Floyd struck eastern North Carolina in September 1999, as many as 50 waste lagoons, many of them several acres in size, were inundated by flood waters. Five lagoons breached, and waterborne animal waste produced nutrient pollution and raised the potential for exposure to pathogens and the risk of disease. As the state recovers, a common question being asked is whether state livestock regulations, particularly regarding facility siting and waste management methods, will be revised to accommodate future flood conditions.