Synthesis of branched antisense oligonucleotides having multiple specificities. Treatment of hormone insensitive prostate cancer.

Antisense oligonucleotides (oligos) directed against transforming growth factor-alpha (TGF-alpha) and its binding site, the epidermal growth factor receptor (EGFR), have demonstrated in vitro and in vivo efficacy against both the PC-3 and LNCaP prostate tumor models. In an attempt to increase the efficiency of these oligos a new type of antisense compound called a bispecific oligo has been evaluated in vitro both alone and in combination with traditional chemotherapeutic agents. These bispecifics, which were first proposed in this journal in 2004, include binding sites for both TGF-alpha and EGFR along the same stretch of complementary DNA. Such bispecifics are able to deliver essentially two antisense activities in an equal molar ratio and can be directed against mRNA encoding proteins of different biochemical pathways. The first bispecifics were developed against two proteins regulating a single autocrine loop. Subsequent bispecifics have been developed which target both EGFR and the apoptosis regulating protein bcl-2. Bispecific activity of a single linear sequence oligo has already been shown to have efficacy. To further develop this multispecific approach, we now propose a branched antisense compound, again, having multiple binding site activities (to complementary sequenced mRNA). Active oligos would be attached to a fat soluble backbone which might enhance targeting and also intracellular entry, release and activity. Such a structure would also permit the customization of these branched forms to include oligos targeting specific proteins related to the growth of various tumor types. Problems associated with the development of antisense oligos have included both membrane solubility and specific targeting. By designing this branched form of antisense structure, multiple activities can be retained (added), solubility improved and delivery enhanced. Such a new formulation would include several antisense oligos covalently bound to and branching off from a lipid-like backbone. An elongated hydrocarbon chain would increase fat solubility and would permit oligo incorporation into nanoparticles or liposome derived delivery vehicles. Specific delivery of oligos could also be enhanced by the tendency of these nanoparticle or liposomal microbubbles to be disrupted under the influence of ultrasonic waves beamed at the targeted tissue.

Caspase-dependent and -independent panc-1 cell death due to actinomycin D and MK 886 are additive but increase clonogenic survival.

In human panc-1 pancreatic cancer cells, actinomycin D (act D) induces a type 1 (apoptotic, extrinsic, death domain, receptor-dependent, and caspase-positive) form of programmed cell death (PCD) and MK 886, a 5-lipoxygenase inhibitor serving among other functions as a surrogate for increasing oxidative stress, a type 2 form, defined as an intrinsic, mitochondria-dependent, autophagic form of cellular suicide. Using both agents simultaneously should allow for examination of their interaction in cells able to express either form of PCD. Activation of both forms might result in synergistic, additive, null, or inhibitory effects on the reduction in proliferation, PCD, and clonogenicity of surviving cells. Co-culture of panc-1 cells with act D and MK 886, which both inhibit their proliferation, had an additive effect on increasing the development of these forms of PCD, as determined by morphology, a nucleosome assay, and flow cytometry. Initially, laddering on agarose detected with propidium iodide, present in act D, and act D plus MK 886-treated cells was partially obscured by randomly degraded DNA. With the use of the more sensitive SYBR green dye and reduced exposure of detached cells to 37 degrees C, a limited laddering of DNA from MK 886-treated cells was also detected. Caspase activity was present in act-D-cultured cells but was absent in cells cultured with MK 886. Combined culture reduced caspase activity in act D-treated cells, consistent with interference from type 2 of type 1 PCD. Removal after 48 hr of act D or MK 886 allowed regrowth of residual cells, the latter agent to a greater extent than the former. In combination, the number of clones was increased compared with act D alone. These features distinguish two forms of PCD. In therapeutic settings in which the modes of cell death have not been identified, unintentional activation of several cellular suicide pathways with "crosstalk" between them occurs. Their intentional simultaneous activation and responses, as modulated by the history of cells in or out of cycle, could reduce the intended therapeutic outcome with survival of additional clonogenic cells due to various forms of mutual interference.

Assessing client satisfaction in a psychology training clinic.

Client satisfaction with services obtained at a clinical psychology training center was assessed. A modified version of the Client Satisfaction Questionnaire was developed to obtain information about levels of satisfaction in such a setting and to evaluate training clinic-specific questions of interest, such as the impact of therapists' experience on satisfaction. Results indicated that client satisfaction is multidimensional. Consistent with other studies, satisfaction was greater among clients who were in therapy for longer periods of time, who completed treatment, and who sought therapy rather than an evaluation. Reasons for dissatisfaction included a wish for more directiveness or advice in therapy and concerns over videotaping procedures. Implications of these results for clinic administrators are discussed.

Children's perceptions of the risk of sexual abuse.

Research to date has not examined children's perceptions of the risk of sexual abuse, and how those perceptions change following participation in a prevention program. This study assessed children's current perceptions of the risk of sexual abuse, changes in children's perceptions of risk after participation in a sexual abuse prevention program, and age differences in children's beliefs about risk. The findings indicate that children's already high perceptions of the risk of sexual abuse increased after participation in a prevention program, and that their predictions of abuse by strangers increased after the program. However, children's predictions of the likelihood of sexual abuse in specific videotaped scenarios was quite low. Younger children had lower perceptions of risk than older children and their perceptions of the likelihood of abuse in the videotaped scenarios remained lower than the perceptions of older children even after the intervention program. In addition, children perceived differences in the risk-levels of various situations, but the factors they used to discriminate risk were not necessarily related to real risks. The findings are discussed in relation to the larger issue of how children's perceptions of the risk of sexual abuse are related to the goals of prevention programs.

AIDS and hemophilia: implications for interventions with families.

Informational needs of hemophiliacs must first be assessed to develop effective educational and prevention programs. A survey of 132 hemophilia patients and family members was conducted to determine the information needs, the preferred source of information and the patients' knowledge of AIDS. Results indicated that the major source of information for the patients was the media; the preferred source was the hemophilia treatment center. Most patients wanted more information about the treatment of AIDS and how to cope with the stresses of AIDS. Although patients indicated that they had received sufficient information about the sexual transmission of AIDS, answers to knowledge questions indicated important misconceptions. Results are discussed with respect to the development of educational and prevention programs for hemophiliacs and their families.