Potential drugs against cervical cancer: zinc-ejecting inhibitors of the human papillomavirus type 16 E6 oncoprotein.

BACKGROUND: The principal agent in the etiology of cervical cancer, i.e., human papillomavirus (HPV) type 16, encodes three oncoproteins, E5, E6, and E7. Structural and mutational studies have identified two potential zinc-finger domains as critical for E6 protein function. We investigated several assays to identify and characterize compounds that interfere with the binding of zinc to E6. METHODS: Thirty-six compounds were selected on the basis of their structure, which would facilitate their participation in sulfhydryl residue-specific redox reactions, and were tested for their ability to release zinc from E6 protein. The zinc-ejecting compounds were then tested for their ability to inhibit E6 binding to E6-associated protein (E6AP) and E6-binding protein (E6BP), two coactivators of E6-mediated cellular transformation. The binding of E6 to E6BP and E6AP was measured by use of surface plasmon resonance (a technique that monitors molecular interactions by measuring changes in refractive index) and by use of in vitro translation assays. The compounds were also tested for their effects on the viability of HPV-containing cell lines. RESULTS: Nine of the 36 tested compounds ejected zinc from E6. Two of the nine compounds inhibited the interaction of E6 with E6AP and E6BP, and one of these two, 4, 4'-dithiodimorpholine, selectively inhibited cell viability and induced higher levels of p53 protein (associated with the induction of apoptosis [programmed cell death]) in tumorigenic HPV-containing cells. CONCLUSION: We have described assay systems to identify compounds, such as 4,4'-dithiodimorpholine, that can potentially interfere with the biology and pathology of HPV. These assay systems may be useful in the development of drugs against cervical cancer, genital warts, and asymptomatic infections by genital HPVs.

Neural differentiation of mesenchymal-like stem cells from cord blood is mediated by PKA.

Mesenchymal stem cells (MSC) are multipotent and give rise to distinctly differentiated cells from all three germ layers. While umbilical cord blood derived mesenchymal-like cells were previously shown to be capable of differentiating into the neural lineage both in vitro and in vivo, the underlying molecular mechanisms and signal transduction pathways remain to be elucidated. In this study, we show that mesenchymal-like cells from umbilical cord blood are capable of neural differentiation and this capability is mediated by the Protein kinase A (PKA) signal transduction pathway. While activation of PKA via experimental cAMP upregulation leads to outgrowth of neurite-like structures as well as expression of neural marker genes, blocking PKA activity completely abolishes all these features. Thus, our results demonstrate that PKA function is sufficient and required for neurite-like outgrowth and regulation of neural specific gene expression in mesenchymal-like stem cells from cord blood.

Biophysical characterization of the oligomeric state of Bax and its complex formation with Bcl-XL.

The overexpression of Bax, a member of the Bcl-2 family, promotes cell death and the dimerization (or oligomerization) of Bax has been shown to be important for its function. Using size-exclusion chromatography and in vitro cross-linking experiments, we demonstrated that Bax exists mainly as a large oligomer of approximately 30 monomeric units. Furthermore, several binding assays demonstrated that Bcl-XL, an anti-apoptotic member of the Bcl-2 family, can bind to the oligomeric form of Bax without requiring Bax to dissociate to monomers.

Truncated hepatitis B virus RNA in human hepatocellular carcinoma: its representation in patients with advancing age.

RNA from tissue samples of 46 HBsAg seropositive hepatocellular carcinoma (HCC) patients was analysed by an RT/PCR assay which discriminates full-length hepatitis B virus (HBV) RNA polyadenylated at the unique viral poly(A) signal governing replication from truncated HBV RNA polyadenylated at a cryptic poly(A) signal. In the tumor the apparent coexistence was less frequent than in the peritumor while the predominance of one of the two RNAs was more frequent. The mean age of patients with a predominance of truncated RNA in the tumor was 9 years above those patients with a predominance of full length RNA (p < 0.05). An inverse relationship existed between the presence of truncated RNA and the presence of RNA carrying core gene sequences. The results of this study establish truncated RNA as a frequent marker of the chronic infection but leave it open whether it is found preferentially in patients developing HCC or generally in chronically infected individuals.

Downregulation of proapoptotic proteins Bax and Bcl-X(S) in p53 overexpressing hepatocellular carcinomas.

As the occurrence of structural p53 mutations in hepatocellular carcinoma (HCC) in Thailand was previously reported to be much lower than that found in other high-incidence HCC areas, we analyzed 16 HCC samples from Thailand to determine the expression and functionality of p53 protein. We observed the overexpression of p53 protein in 69% of HCC, despite the prevalence of the wild-type p53 gene. However, the overexpressed p53 protein was nonfunctional as suggested by its inability to modulate the expressions of several p53 effector proteins (p21 and Bcl-2 family proteins). In addition, we observed significant underexpression of two proapoptotic proteins, Bax and Bcl-X(S), in 81% (P = 0.02) and 64% (P = 0.03) of HCC, respectively. Consequently, the ratios of proapoptotic to antiapoptotic BCL-2 family proteins were reduced in 88% of the HCC tumor tissues when compared to normal tissues, such that the rheostat between BCL-2 family proteins is strongly skewed toward enhanced cell survival in the tumor cells.

Inactivation of the human papillomavirus-16 E6 oncoprotein by organic disulfides.

We are investigating compounds that could be useful in the treatment of neoplastic lesions of the cervix by acting on the oncoprotein E6 of human papillomavirus-16. The E6 protein contains two potential zinc-binding domains that are required for most of its functions. We have published tests that measure (i) the release of zinc ions after chemical alteration of the cysteine groups of these zinc-binding domains (TSQ assay), (ii) the interaction of E6 with the cellular proteins E6AP and E6BP (BIACORE assay), and (iii) the viability of tumor cell lines that require the continuous expression of HPV oncoproteins (WST1 assay). Based on these tests, we identified 4.4'-dithiodimorpholine as a potential lead compound. In this study we examined whether the dithiobisamine moiety of 4,4'-dithiodimorpholine may be an important molecular prerequisite for further drug development in this system. We have evaluated 59 new substances including organic disulfides and those containing the dithiobisamine moiety, as well as structural analogues. The compounds with significant reactivity in all three assays were observed only for dithiobisamine derivatives with saturated cyclic amines and aryl substituted piperazines. The identity of these substances suggests that the N-S-S-N moiety is necessary but not sufficient for reactivity in our assays, and that dithiobisamine based substances are useful as lead compounds that target the cysteine groups of HPV-16 E6 zinc fingers.

Downregulation of beta2-microglobulin in human cord blood somatic stem cells after transplantation into livers of SCID-mice: an escape mechanism of stem cells?

Adherently growing, non-hematopoietic somatic stem cells isolated from human cord blood were stained with the fluorescent dye PKH26 and transplanted into livers of SCID-mice to examine a possible cell fate transition. Already 7 days after transplantation stem cells were well integrated into the liver tissue. Human albumin that was not expressed by the stem cells before transplantation was detectable in the host's livers after injection of cord blood stem cells. Human alpha1-antitrypsin was detectable in stem cells already before transplantation and remained positive in the mouse liver. The most interesting observation in this study was the downregulation of human beta2-microglobulin (beta2M) in the stem cells after transplantation: beta2M is expressed constitutively in our cord blood stem cells. However, beta2M was no longer detectable by RT-PCR in all tissues where human albumin and alpha1-antitrypsin were expressed after stem cell transplantation. beta2M is known to participate as an integral part of the major histocompatibility complex. Absence of beta2M makes the residual heavy chain inactive as an antigen. Thus, downregulation of beta2M may represent an escape mechanism from killer-T cells and may be a molecular mechanism explaining the recently described "immunological blindness" [37] of stem cells. In contrast to the results obtained after direct injection of stem cells as a suspension, no consistent downregulation of beta2M was observed after transplantation of stem cells encapsulated in alginate beads to generate a compartment where stem cells are protected from the host's natural killer cells. No expression of human genes was observed after transplantation of human cord blood derived mononuclear cells (MNC) that were used as a negative control. In conclusion, we have shown that human cord blood somatic stem cells survive and are reprogrammed after transplantation into mouse livers, although a complete transdifferentiation to hepatocytes did not occur within 7 days, since some marker genes (GATA4 and alpha-fetoprotein) were still negative. Switching off expression of beta2M may be part of an intriguing and novel mechanism explaining why stem cells escape the host's immune system.