Molecular characterization of photosensitizer-mediated photodynamic therapy by gene expression profiling.

Photodynamic therapy (PDT) is a novel cancer treatment based on the tumor-specific accumulation of a photosensitizer followed by irradiation with visible light, which induces selective tumor cell death via production of reactive oxygen species. To elucidate the underlying mechanisms, microarray analysis was used to analyze the changes in gene expression patterns during PDT induced by various photosensitizers. Cancer cells were subjected to four different photosensitizer-mediated PDT and the resulting gene expression profiles were compared. We identified many differentially expressed genes reported previously as well as new genes for which the functionfunctions in PDT are still unclear. Our current results not only advance the general understanding of PDT but also suggest that distinct molecular mechanisms are involved in different photosensitizer-mediated PDT. Elucidating the signaling mechanisms in PDT will provide information to modulate the antitumor effectiveness of PDT using various photosensitizers.

Clinical presentations and skin denervation in amyloid neuropathy due to transthyretin Ala97Ser.

OBJECTIVE: Familial amyloid polyneuropathy (FAP) due to amyloidogenic transthyretin (TTR) is often associated with impairment of thermonociceptive functions. This study investigated skin innervation and its clinical significance in genetically defined FAP due to a hot-spot Ala97Ser TTR mutation (Ala97Ser). METHODS: Skin biopsies were performed on the distal leg of patients with Ala97Ser, and intraepidermal nerve fiber (IENF) densities were quantified. RESULTS: There were 19 unrelated patients with Ala97Ser manifesting a late-onset (59.47 +/- 5.70 years) generalized neuropathy with disabling motor, sensory, and autonomic symptoms. Against a background of a slowly progressive course, 7 patients (36.8%) exhibited additional rapid declines in neurologic deficits, which were associated with elevation of the protein content in the CSF (p < 0.001). The IENF density was markedly reduced in Ala97Ser patients compared to age- and gender-matched controls (0.99 +/- 1.11 vs 8.31 +/- 2.87 fibers/mm, p < 0.001). Skin denervation was present in all patients and was lower in patients with a higher disability grade (0.17 +/- 0.26 vs 1.37 +/- 1.16 fibers/mm, p = 0.003). Albuminocytologic dissociation in the CSF was observed in 14 patients (73.7%), and the IENF density was negatively correlated with the CSF protein concentration (p = 0.015). CONCLUSIONS: Skin denervation was common in Ala97Ser, and degeneration of cutaneous nerve terminals was correlated with the severity of clinical phenotypes and the level of CSF protein.

Roles of the AX(4)GKS and arginine-rich motifs of hepatitis C virus RNA helicase in ATP- and viral RNA-binding activity.

The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) possesses protease, nucleoside triphosphatase, and helicase activities. Although the enzymatic activities have been extensively studied, the ATP- and RNA-binding domains of the NS3 helicase are not well-characterized. In this study, NS3 proteins with point mutations in the conserved helicase motifs were expressed in Escherichia coli, purified, and analyzed for their effects on ATP binding, RNA binding, ATP hydrolysis, and RNA unwinding. UV cross-linking experiments indicate that the lysine residue in the AX(4)GKS motif is directly involved in ATP binding, whereas the NS3(GR1490DT) mutant in which the arginine-rich motif (1486-QRRGRTGR-1493) was changed to QRRDTTGR bound ATP as well as the wild type. The binding activity of HCV NS3 helicase to the viral RNA was drastically reduced with the mutation at Arg1488 (R1488A) and was also affected by the K1236E substitution in the AX(4)GKS motif and the R1490A and GR1490DT mutations in the arginine-rich motif. Previously, Arg1490 was suggested, based on the crystal structure of an NS3-deoxyuridine octamer complex, to directly interact with the gamma-phosphate group of ATP. Nevertheless, our functional analysis demonstrated the critical roles of Arg1490 in binding to the viral RNA, ATP hydrolysis, and RNA unwinding, but not in ATP binding.

Color Doppler vascularity index can predict distant metastasis and survival in colon cancer patients.

The purpose of this study was to investigate the clinical usefulness of the color Doppler vascularity index (CDVI) in patients with colon cancer before surgery. Forty-four patients with sonographically visible tumor mass of colon cancer were investigated. The CDVI of each tumor was determined using transabdominal color Doppler ultrasound. The CDVI was defined as the ratio of the number of the colored pixels within a tumor section to the number of total pixels in that specific tumor section and was calculated by using Encomate software (Electronic Business Machine Co. Ltd., Taipei, Taiwan). The correlation between the CDVI and clinicopathological factors, mode of recurrence, and patient survival was studied. For comparison, microvessel density (the mean number of microvessels in three areas of highest vascular density at x200 magnification) of the tumors of these 44 patients was also evaluated by using immunohistochemical staining of surgical specimens with anti-CD34. The microvessel density was not correlated with Dukes' classification, clinicopathological factors, and survival. The CDVI was significantly higher in the patients with lymph node metastases and vascular invasion than in those without such metastases and invasion (P = 0.006 and P = 0.0098, respectively). Moreover, in patients with a high CDVI (> 15%) and positive vascular invasion, survival was significantly poorer than in those with low CDVI (< or = 15%) and negative invasion (P = 0.0037 and 0.0039, respectively). Multivariate analysis indicated that liver metastasis, vascular invasion, and CDVI are independent prognostic factors in the patients with colon cancer. According to the mode of recurrence in 36 patients who underwent curative resection, the frequency of the distant organ recurrence was significantly higher in the high CDVI group (40%) than in the low CDVI group (0%). The CDVI is a good preoperative indicator of recurrence and patient survival in colon cancer. Thus, the CDVI may be helpful in stratifying patients for adjuvant therapy.

A 12-amino acid stretch in the hypervariable region of the spike protein S1 subunit is critical for cell fusion activity of mouse hepatitis virus.

The spike (S) glycoprotein of mouse hepatitis virus (MHV) plays a major role in the viral pathogenesis. It is often processed into the N-terminal S1 and the C-terminal S2 subunits that were evidently important for binding to cell receptor and inducing cell-cell fusion, respectively. As a consequence of cell-cell fusion, most of the naturally occurring infections of MHV are associated with syncytia formation. So far, only MHV-2 was identified to be fusion-negative. In this study, the S gene of MHV-2 was molecularly cloned, and the nucleotide sequence was determined. The MHV-2 S protein lacks a 12-amino acid stretch in the S1 hypervariable region from amino acid residue 446 to 457 when compared with the fusion-positive strain MHV-JHM. In addition, there are three amino acid substitutions in the S2 subunit, Tyr-1144 to Asp, Glu-1165 to Asp, and Arg-1209 to Lys. The cloned MHV-2 S protein exhibited the fusion-negative property in DBT cells as the intrinsic viral protein. Furthermore, similar to the fusion-positive MHV-JHM strain, proteolytic cleavage activity was detected both in DBT cells infected with the fusion-negative MHV-2 and in the transfected cells that expressed the cloned MHV-2 S protein. Domain swapping experiments demonstrated that the 12-amino acid stretch missing in the MHV-2 S1 subunit, but not the proteolytic cleavage site, was critical for the cell-fusion activity of MHV.

Prognostic factors in patients with bulky stage IB or IIA cervical carcinoma undergoing neoadjuvant chemotherapy and radical hysterectomy.

All patients with bulky (> or =4 cm) Stage Ib or IIa cervical carcinoma treated at Chang Gung Memorial Hospital between August 1988 and December 1991 using a strategy of neoadjuvant chemotherapy with cisplatin, vincristine, and bleomycin and radical hysterectomy were reviewed. Fifty-nine evaluable patients received 1 to 3 courses of chemotherapy, and 51 underwent subsequent hysterectomy. The remaining 8 patients, not completing planned surgery, were treated with definitive radiotherapy. The overall clinical response rate was 81.4% (48/59) with 18.6% complete response. Clinical response to chemotherapy was not different by stage, histologic type, tumor size, level of squamous cell carcinoma antigen, or DNA ploidy. However, tumors with DNA indices (DI) greater than 1.3 were associated with higher clinical response rates than tumors with DI < or = 1.3 (P = 0.043). Histologically proven pelvic node metastases was noted in 18.5% (10/54) who had laparotomy. Concomitant pregnancy and more than one node metastases had significant adverse influence on recurrence and death. The 5-year survival rate of those patients who received hysterectomy was 80.3%, while only 1 of the 8 patients without hysterectomy survived. Of the 7 patients received hysterectomy despite clinical poor response, only 2 had node metastases and 3 died, whereas all the 4 patients deterred hysterectomy for poor response died. This study demonstrates the value of DNA flow cytometry in predicting chemosensitivity. However, with a DI cutoff at 1.3, only 29.2% patients could be selected. Further studies are necessary to find additional indicators that predict histological response to select better candidates for this approach and to determine optimal adjunctive treatment in case that poor prognostic features are found.

Role of the electrostatic attractive force in the adsorption of proteins by aluminum hydroxide adjuvant.

The fact that both aluminum hydroxide adjuvant and proteins have a pH dependent surface charge means that electrostatic forces play a role in the adsorption of proteins by aluminum hydroxide adjuvant during the preparation of vaccines. The objective of this study was to examine the contribution of the electrostatic attractive force in the adsorption of proteins by aluminum hydroxide adjuvant. Since the surface charge characteristics of aluminum hydroxide adjuvant can be modified by the adsorption of phosphate anion, a series of aluminum hydroxide adjuvants were prepared by treatment with various concentrations of phosphate anion. The isoelectric points (iep) of these adjuvants ranged from 11.0 to 4.6 and the electrophoretic mobilities at pH 7.4 ranged from 2.0 to -3.3 microns cm/V s. The line broadening of the (020) band of the X-ray diffraction pattern indicated that treatment with phosphate anion did not change the primary crystallite dimension. Adsorption at pH 7.4 of positively charged lysozyme (iep = 11.1) was directly related to the negative surface charge of the adjuvant. No adsorption occurred when the surface charge was positive. In contrast, negatively charged ovalbumin (iep = 4.6) was adsorbed by all of the adjuvants at pH 7.4, although the adsorptive capacity was the greatest when the surface charge was positive. The results indicate that adsorptive forces in addition to the electrostatic attractive force play an important role in the adsorption of some proteins by aluminum hydroxide adjuvant. It is believed the structurally flexible proteins, like ovalbumin, exhibit more complex adsorption behavior than structurally rigid proteins, like lysozyme, for which adsorptive behavior can be explained by electrostatic forces.

Breast cancer vascularity: color Doppler sonography and histopathology study.

In this prospective study, the authors examined 50 patients with breast tumors (malignant, n = 32; benign, n = 18) to investigate the correlation between color Doppler flow mapping and histopathological findings and to evaluate the clinical significance of color Doppler mapping. Among the 32 patients with breast cancer, color Doppler signals were detected in 24 patients (75%). The maximum flow velocities varied from 5 to 34 cm/sec, with 16 (67%) of them above 15 cm/sec. Among the 18 patients with benign tumors, color Doppler signals could be detected in 7 (39%). The maximum flow velocity varied from 3 to 30 cm/sec but was over 15 cm/sec in only two patients (28%). Histological studies revealed that color Doppler signals detected by Doppler sonography correlated with disordered neovascularization penetrating the lesion from its periphery, consisting of thin-walled blood vessels and large arteriovenous shunts. Although large tumors tend to have high Doppler flow, there is no significant correlation between the maximum flow velocity and tumor size. There is also no significant correlation between the detection of high flow color Doppler signals and the age, receptor status, tumor size, lymph node metastases, or clinical stage of patients with breast cancer. However, there is a positive association (p < 0.05) between nodal metastases and higher tumor flow velocity in T1 (< or = 2 cm) breast tumors, but not in larger tumors. It is concluded that color Doppler is useful in the assessment of tumor vascularity but is of limited value in the differentiation of benign from malignant lesions. However, the presence of color Doppler signals in T1 breast cancer suggesting early dissemination of the cancer might be of important clinical significance in detecting those small, apparently early, but aggressive tumors with poor prognosis.

Functional domains of delta antigens and viral RNA required for RNA packaging of hepatitis delta virus.

The functions of delta antigens (HDAgs) in the morphogenesis of hepatitis delta virus (HDV) have been studied previously. The C terminus of large HDAg has been shown to complex with the small surface antigen (HBsAg) of helper hepatitis B virus, whereas the assembly of small HDAg requires interaction with the N terminus of large HDAg (M.-F. Chang, C.-J. Chen, and S. C. Chang, J. Virol. 68:646-653, 1994). To further examine the molecular mechanisms by which HDAgs are involved in the assembly of HDV RNA, we have cotransfected Huh-7 cells with plasmids representing a longer than unit-length HDV and the small HBsAg cDNAs. We found that HDAg mRNA could be generated from an endogenous promoter within the HDV cDNA that was translated into large HDAg. Large HDAg is capable of complexing with monomeric HDV genomic RNA to form ribonucleoprotein particles (RNPs) and is capable of forming enveloped HDV-like particles in the presence of small HBsAg without undergoing HDV replication. In addition, the middle region from amino acid residues 89 to 145 of large HDAg is required for assembly of the RNPs but is dispensable for assembly of the enveloped particles. RNA assembly is also demonstrated with small HDAg when it is cotransfected with a packaging-defective large HDAg mutant and small HBsAg. Leu-115 within the putative helix-loop-helix structure of the small HDAg is important for the replication of HDV but is not essential for RNA assembly, suggesting that conformational requirements of small HDAg for replication and assembly of viral RNA may be different. Further studies indicate that a 312-nucleotide linear HDV RNA from one end of the HDV and structure is sufficient to form RNP complexes competent for assembly of virus-like particles with large HDAg and small HBsAg.

Cellular proteins specifically bind to the 5'-noncoding region of hepatitis C virus RNA.

Hepatitis C virus (HCV) RNA contains a highly conserved 5'-noncoding region (5'NCR) which may be important in viral multiplication. To study the possible mechanisms of the cellular proteins involved in HCV replication and pathogenesis, a gel mobility shift assay and competition analysis were performed with the HCV 5'NCR. Two specific complexes were formed between the 341-nucleotide RNA of the HCV 5'NCR and proteins of mammalian cells. The specific RNA-protein complexes were maintained in the region of the 5'NCR from nucleotides 131 to 253. Nevertheless, the slower migrating RNA-protein complex failed to form when a polypyrimidine tract sequence (191-UCCUUUCUU-199) in the stem-loop III structure of HCV 5'NCR was changed to 191-UCCUUUggU-199. A uv cross-linking assay further identified two cellular proteins, p87 and p120, that specifically bound to the stem-loop III structure. Mutations at the polypyrimidine tract sequence inhibited the binding of p87, but maintained the ability of the mutant HCV RNA to interact with p120. Translation competition assay demonstrated that the 5'NCR from nt 131 to 253 within the stem-loop III structure is important for the translation of HCV core protein. In addition, p120 and unidentified cellular proteins are likely to be involved in the translation of HCV polyprotein, whereas p87 may play important roles in HCV multiplication other than translation.

Nuclear localization signals in the core protein of hepatitis C virus.

The core protein of the hepatitis C virus is derived from the N-terminal 191 amino acids of the viral polyprotein by proteolytic cleavage. In the current study, subcellular localizations of the HCV core and its beta-galactosidase fusion proteins in transfected cells were examined by indirect immunofluorescence and cytochemical staining. The core protein was located predominantly in the cytoplasm 6 days after a plasmid encoding the full-length core protein had been introduced into mammalian cells. A hydrophobic domain in the C-terminal region of the core protein may block the efficiency of nuclear transport, since a beta-galactosidase fusion protein that contains HCV core protein lacking the C-terminal 66-amino-acid was located within the nuclei of mammalian cells 24 hours posttransfection. Three independent nuclear localization signals were further identified in the N-terminal region of the HCV core protein.

The uses and limitations of DNA flow cytometry in stage IB or II cervical carcinoma.

BACKGROUND: The role of flow cytometry in predicting prognosis for cervical carcinoma remains unclear. METHODS: Flow cytometric analysis was performed on tissues, fixed in formaldehyde solution and embedded in paraffin, from 411 patients with Stage IB or II cervical carcinoma who had been treated with radical abdominal hysterectomy and bilateral pelvic lymphadenectomy. RESULTS: DNA aneuploid-multiploid tumors were found in 37.5%, tetraploid in 4.6%, and diploid-peridiploid in 57.9%. Five-year recurrence-free survival rates of the three groups were 74.3%, 77.8%, and 76.4%, respectively (P > 0.05). DNA aneuploidy and DNA index (DI) of greater than 1.3 were highly correlated to parametria extension. In univariate analysis, pelvic lymph node metastases, stage, parametrial extension, depth of cervical stromal invasion, tumor size, and DI (1.3, 1.4, 1.5 as breakpoint) were significant prognostic factors. DNA ploidy, S-phase fraction, and S-G2M fraction were not significant. In multivariate analysis, DI of greater than 1.3, pelvic node metastases, clinical Stage II, and depth of stromal invasion greater than two-thirds of full cervical thickness were independent and significant variables. The prognostic index (PI), defined by the model, was able to categorize the patients into three distinct risk groups. The 5-year recurrence free survival rates of the low-, intermediate-, and high-risk groups were 89.5%, 73.0%, and 58.9%, respectively (P < 0.0001). CONCLUSIONS: The prognostic value of the DI as a single variable is promising and warrants additional investigation to establish its appropriate use.

Nuclear localization signals, but not putative leucine zipper motifs, are essential for nuclear transport of hepatitis delta antigen.

Hepatitis delta antigen (HDAg) is the only known protein of hepatitis delta virus and was previously shown to localize in the nucleoplasm of infected liver cells. In this study, nuclear localization signals of HDAg were defined by expressing various domains of the antigen in both hepatic and nonhepatic cells as beta-galactosidase fusion proteins. A cytochemical staining assay demonstrated that a domain from amino acid residues 35 to 88 of HDAg was able to facilitate transport to the nucleus of the originally cytoplasm-localized protein beta-galactosidase. Two nuclear localization signals, NLS1 and NLS2, which are similar to those of simian virus 40 T antigen and polyomavirus T antigen, respectively, were identified. Either NLS1 or NLS2 alone was sufficient for the nuclear transport of HDAg. However, a fusion protein (N65Z) containing beta-galactosidase and the N-terminal 65 amino acids of HDAg, containing NLS1, was localized exclusively in the cytoplasm and perinuclear region. A possible hydrophobic subdomain between amino acid residues 50 and 65 may block the function of NLS1. Nevertheless, N65Z could enter the nuclei of transfected cells when it was coexpressed with full-length HDAg. Entry into the nucleus may be mediated by the coiled-coil structure rather than the putative leucine zipper motif located between amino acid residues 35 and 65. The existence of two independent nuclear localization signals may ensure the proper functioning of HDAg in the multiplication of delta virus in the nucleus. In addition, two putative casein kinase II sites (SRSE-5 and SREE-126) that may be important in controlling the rate of nuclear transport were found in HDAg.

Identification of a domain required for autoproteolytic cleavage of murine coronavirus gene A polyprotein.

The 5'-most gene of the murine coronavirus genome, gene A, is presumed to encode viral RNA-dependent RNA polymerase. It has previously been shown that the N-terminal portion of this gene product is cleaved into a protein of 28 kilodaltons (p28). To further understand the mechanism of synthesis of the p28 protein, cDNA clones representing the 5'-most 5.3 kilobases of murine coronavirus mouse hepatitis virus strain JHM were sequenced and subcloned into pT7 vectors from which RNAs were transcribed and translated in vitro. The sequence was found to encode a single long open reading frame continuing from near the 5' terminus of the genome. Although p28 is encoded from the first 1 kilobase at the 5' end of the genome, translation of in vitro-transcribed RNAs indicated that this protein was not detected unless the product of the entire 5.3-kilobase region was synthesized. Translation of RNAs of 3.9 kilobases or smaller yielded proteins which contained the p28 sequence, but p28 was not cleaved. This suggests that the sequence in the region between 3.9 and 5.3 kilobases from the 5' end of the genomic RNA is essential for proteolytic cleavage and contains autoproteolytic activity. The p28 protein could not be cleaved from the smaller primary translation products of gene A, even in the presence of the larger autocleaving protein. Cleavage of the p28 protein was inhibited by addition of the protease inhibitor ZnCl2. This study thus identified a protein domain essential for autoproteolytic cleavage of the gene A polyprotein.