Demonstration of wavelength-scan-free action spectroscopy in pump/probe measurement with supercontinuum pump light.

We devise and introduce the principle of wavelength-scan-free spectroscopy for the pump light in pump/probe measurement (action spectroscopy) using supercontinuum light; we demonstrate its implementation by measuring transmission spectra. We use the supercontinuum light noise as a code in order to discriminate wavelength. We extract the stimulation at the desired wavelength by correlating the noise at that wavelength observed separately and the observed total stimulation carried by the probe light. The wavelength-scan-free spectroscopy is enabled with a simultaneous procedure for multiple wavelengths.

Fluorescence anisotropy in indole under two-photon excitation in the spectral range 385-510 nm.

This paper presents the detailed study of two-photon excited fluorescence in indole dissolved in propylene glycol produced by two-photon absorption from the molecular ground state to several high lying excited states. The experimental method involved excitation with linearly and circularly polarized femtosecond pulses and time-resolved detection of the polarized fluorescence decay. The fluorescence intensity, anisotropy, excited state lifetime, and rotation diffusion time as function of the excitation light wavelength in the spectral range 385-510 nm were determined in experiment. The theoretical fit of the experimental results obtained demonstrated the contributions of six highly excited molecular states of different symmetry to the two-photon absorption intensity and fluorescence anisotropy. An intense two-photon absorption peak was observed experimentally in the spectral range 385-480 nm and explained as contributions from four high lying electronic excited states. The temporal dependence of fluorescence intensity in indole was satisfactory characterized by a single excited state lifetime τf and a single rotational diffusion time τrot. As shown, the excited state lifetime τf depends on the excitation light wavelength, which was explained by taking into account nonradiative relaxation transitions in the molecular vibronic excited states. The rotation diffusion time τrot was found to be equal to τrot = 0.9 ± 0.5 ns and practically independent of the excitation wavelength. The determined molecular anisotropy changed substantially in the spectral area 385-480 nm taking positive and negative values, and the anisotropies referring to linearly and circularly polarized excitation light changed almost in opposite phases with each other. The experimental results obtained were interpreted using ab initio molecular structure computations and a model based on the Frank-Condon approximation and taking into account vibronic absorption bands.

Outcomes of fulvestrant therapy among japanese women with advanced breast cancer: a retrospective multicenter cohort study (JBCRG-C06; Safari).

PURPOSE: This retrospective study evaluated the effect of clinical background and treatment line on time to treatment failure (TTF) in advanced/metastatic breast cancer (AMBC) patients receiving F500 in Japan (UMIN 000015168). METHODS: Patients who commenced F500 treatment were registered at 16 sites in Japan. Correlations between baseline clinicopathological factors, treatment line, and TTF were investigated by Kaplan-Meier analysis. TTF data were analyzed using univariate analysis and multivariate analysis with a Cox proportional hazards model. RESULTS: Data for 1072 patients were available; 1031 patients (96.2%) were evaluable for efficacy. F500 was administered as first-line treatment in 2.0%, second-line in 22.7%, third-line in 26.7%, and ≥fourth-line in 48.6% patients. Median TTF was 5.4 months. Multivariate analysis found that earlier F500 use (first and second vs. third vs. ≥fourth line; hazard ratio (HR) = 0.80, 95% confidence interval (CI) 0.74-0.86; P < 0.001), longer period from AMBC diagnosis to F500 use (≥3 vs. <3 years; HR 0.60, 95% CI 0.51-0.70; P < 0.001), and no prior palliative chemotherapy administered for unresectable or metastatic breast cancer (no vs. yes; HR 0.69, 95% CI 0.60-0.80; P < 0.001) were associated with significantly longer TTF. Among 691 patients, where information on histologic/nuclear grade was available, a low grade was also associated with a longer TTF, but this finding was not maintained among patients with recurrent breast cancer (N = 558). Among women with recurrent breast cancer, a longer DFI between a patient's initial breast cancer diagnosis and their recurrence was associated with a longer TTF on F500 therapy. CONCLUSIONS: Our study showed that treatment period of F500 was longer when used in earlier-line treatment. For patients on F500, TTF was also longer for patients who had not received prior palliative chemotherapy and for those who had a longer period from their AMBC diagnosis to F500 use.

Retrospective study to estimate the prevalence and describe the clinicopathological characteristics, treatments received, and outcomes of HER2-low breast cancer.

BACKGROUND: Approximately 80% of all breast cancers (BCs) are currently categorized as human epidermal growth factor receptor 2 (HER2)-negative [immunohistochemistry (IHC) 0, 1+, or 2+/in situ hybridization (ISH) negative]; approximately 60% of BCs traditionally categorized as HER2-negative express low levels of HER2. HER2-low (IHC 1+ or IHC 2+/ISH-) status became clinically actionable with approval of trastuzumab deruxtecan to treat unresectable/metastatic HER2-low BC. Greater understanding of patients with HER2-low disease is urgently needed. PATIENTS AND METHODS: This global, multicenter, retrospective study (NCT04807595) included tissue samples from patients with confirmed HER2-negative unresectable/metastatic BC [any hormone receptor (HR) status] diagnosed from 2014 to 2017. Pathologists rescored HER2 IHC-stained slides as HER2-low (IHC 1+ or IHC 2+/ISH-) or HER2 IHC 0 after training on low-end expression scoring using Ventana 4B5 and other assays at local laboratories (13 sites; 10 countries) blinded to historical scores. HER2-low prevalence and concordance between historical scores and rescores were assessed. Demographics, clinicopathological characteristics, treatments, and outcomes were examined. RESULTS: In rescored samples from 789 patients with HER2-negative unresectable/metastatic BC, the overall HER2-low prevalence was 67.2% (HR positive, 71.1%; HR negative, 52.8%). Concordance was moderate between historical and rescored HER2 statuses (81.3%; κ = 0.583); positive agreement was numerically higher for HER2-low (87.5%) than HER2 IHC 0 (69.9%). More than 30% of historical IHC 0 cases were rescored as HER2-low overall (all assays) and using Ventana 4B5. There were no notable differences between HER2-low and HER2 IHC 0 in patient characteristics, treatments received, or clinical outcomes. CONCLUSIONS: Approximately two-thirds of patients with historically HER2-negative unresectable/metastatic BC may benefit from HER2-low-directed treatments. Our data suggest that HER2 reassessment in patients with historical IHC 0 scores may be considered to help optimize selection of patients for treatment. Further, accurate identification of patients with HER2-low BC may be achieved with standardized pathologist training.

Sirt1 inhibitor, Sirtinol, induces senescence-like growth arrest with attenuated Ras-MAPK signaling in human cancer cells.

The induction of senescence-like growth arrest has emerged as a putative contributor to the anticancer effects of chemotherapeutic agents. Clinical trials are underway to evaluate the efficacy of inhibitors for class I and II histone deacetylases to treat malignancies. However, a potential antiproliferative effect of inhibitor for Sirt1, which is an NAD(+)-dependent deacetylase and belongs to class III histone deacetylases, has not yet been explored. Here, we show that Sirt1 inhibitor, Sirtinol, induced senescence-like growth arrest characterized by induction of senescence-associated beta-galactosidase activity and increased expression of plasminogen activator inhibitor 1 in human breast cancer MCF-7 cells and lung cancer H1299 cells. Sirtinol-induced senescence-like growth arrest was accompanied by impaired activation of mitogen-activated protein kinase (MAPK) pathways, namely, extracellular-regulated protein kinase, c-jun N-terminal kinase and p38 MAPK, in response to epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I). Active Ras was reduced in Sirtinol-treated senescent cells compared with untreated cells. However, tyrosine phosphorylation of the receptors for EGF and IGF-I and Akt/PKB activation were unaltered by Sirtinol treatment. These results suggest that inhibitors for Sirt1 may have anticancer potential, and that impaired activation of Ras-MAPK pathway might take part in a senescence-like growth arrest program induced by Sirtinol.

Cleavage of human serum immunoglobulin G by an immobilized pepsin preparation.

In order to obtain an efficacious and safe immunoglobulin G (IgG) preparation for intravenous use, the digestion of IgG with an immobilized pepsin (EC 3.4.23.1) preparation was studied. Thus, pepsin was immobilized onto glutaraldehyde-activated AH-Sepharose 4B under acidic conditions. THe enzymatic properties, such as proteolytic activity, pH-activity profile and heat stability, of the immobilized pepsin preparation were examined. The immobilized pepsin retained more than 40% of its proteolytic activity toward N-acetyl-L-phenylalanyl-L-3,5-diiodo-tyrosine and more than 30% toward IgG, and also remarkable stability as compared with free pepsin. The immobilized pepsin thus prepared was efficiently used for the limited cleavage of IgG and the gel-filtration effect of the column made it easily possible to yield the F(ab')2-rich fraction for intravenous use.

Identification of lysine-238 of Escherichia coli biotin carboxylase as an ATP-binding residue.

Escherichia coli biotin carboxylase was affinity labeled with adenosine diphosphopyridoxal to identify its ATP binding site. Lysyl endopeptidase digestion of the modified protein, followed by high performance liquid chromatography separation and amino acid sequencing allowed to identify lysine-238 to be the site of modification. Site-directed mutagenesis of this residue into alanine, arginine or glutamine resulted in mutants with much decreased activity. Lysine-238 seems to interact with the gamma-phosphate group of ATP but is not involved in catalysis.

Differential growth inhibition by 5-fluorouracil in human colorectal carcinoma cell lines.

The effects of 5-fluorouracil (5-FU) on cell growth were investigated using a primary culture of human fibroblasts, MRC-5, and three established human colon cancer cell lines, DLD-1, LoVo and SW620. Detailed flow cytometric analyses revealed differential growth inhibition among these cell lines including three modes of cell growth modulation: (a) loss or accumulation of S phase cells; (b) G2/M block; and (c) G1-S arrest. From analyses on the amount of 5-FU incorporated into cellular RNA and the activity of thymidylate synthase (TS), suppression of TS and depletion of dTTP, a possible consequence of the former, was considered to be the major action of 5-FU in these cells. Differences in the cellular responses to the nucleotide pool imbalance appeared to make the cell growth modulation diverse. Loss of S phase cells and G1-S phase arrest were evident in p53 wild-type cells, MRC-5 and LoVo. Cells proficient in DNA mismatch repair, SW620 and MRC-5, showed marked modulations in S-G2/M progression. These findings suggest that multiple factors, including p53 and DNA mismatch repair, participate in diverse cell growth modulations in cells treated with 5-FU. Cellular resistance to 5-FU correlated well with a loss of modulations in S-G2/M progression, rather than with a defect of G1-S arrest, which suggests the significance of DNA mismatch repair as a factor affecting the sensitivity of cells to 5-FU.

Reduced expression of p33(ING1) and the relationship with p53 expression in human gastric cancer.

p33(ING1) is a novel growth inhibitor candidate for a tumor suppressor gene. p33(ING1) cooperates with p53 and negatively regulates cell growth by activating transcription from the p21/WAF1 promoter even though it has no significant sequence similarity to p53. We first compared p33(ING1) expression in human gastric cancers and matched normal tissues using quantitative RT-PCR and real time 'Taqman TM' technology. A significant decrease in p33(ING1) expression was evident in 15 of 20 gastric cancers. In immunohistochemical analysis, p53 protein expression was detected in 4 of 20 (20%) tumors, and 12 of 15 (80%) tumors with decreasing p33(ING1) expression in RT-PCR had the wild type p53. When we examined the sequence of p33(ING1) in 12 gastrointestinal carcinoma cell lines, we found mutation in only one cell line, HCT116. Our findings are interpreted to mean that p33(ING1) may function as a tumor suppressor in gastric carcinogenesis, even though the gene is preserved in the majority of gastrointestinal carcinomas. It should be noted that expression of p33 decreased in many cancer patients, and the biological effects of p33(ING1) and p53 are interrelated and require the activity of both genes.

Diminished expression of ING1 mRNA and the correlation with p53 expression in breast cancers.

p33(ING1) is a novel candidate tumor suppressor and its overexpression induces growth arrest or apoptosis in different cell lines. These functions of p33(ING1) depend largely on the activity of p53, and p53-dependent activation of the transcription from the p21/WAF1 promoter also requires p33(ING1). We examined the expression of ING1 mRNA in breast cancer cell lines and clinical breast cancer tissues, using quantitative RT-PCR and real time TaqMan technology. In breast cancer cell lines, ING1 mRNA was expressed at almost the same level. However, in a comparison between the cancer and matched normal tissues, a significant decrease in ING1 mRNA expression was found in 17 of 24 (70.8%) breast cancer tissues. We also examined the correlation between ING1 mRNA expression and p53 expression. There was a significant decrease of ING1 mRNA in nine of 15 tumors negative for p53 immunostaining, most of which were considered to have wild type p53. In these tumors, p53 may not function in case of a decreased expression of p33(ING1), and the lack of cell cycle regulation may correlate with the carcinogenesis and tumor progression.

Vascular invasion and potential for tumor angiogenesis and metastasis in gastric carcinoma.

BACKGROUND: Hematogenous metastasis occurs when cancer cells released from the primary site enter blood vessels and are transported to distant organs, where they attach and proliferate. Angiogenesis is essential for tumor growth and metastasis and depends on the production of angiogenic factors by tumor cells. METHODS: We analyzed data on 1184 Japanese adult men and women with gastric cancer with respect to the relation between vascular invasion and the potential for tumor angiogenesis and metastasis. All these patients were treated from 1976 to 1995 in the Department of Surgery II, Kyushu University. In 300 patients, the expression of vascular endothelial growth factor (VEGF) and p53 protein in tumor tissues was examined by using an immunohistochemical staining method or Northern blotting or both. Intratumoral microvessels were stained with anti-CD31 monoclonal antibody. RESULTS: Vascular invasion was evident in 254 patients (21.5%), and in these patients lymphatic invasion was more frequent and the rate of lymph node metastasis was higher in relation to the extent of vascular invasion. The positive findings were directly related to the depth of invasion and the presence of lymph node and liver metastasis. Tumor invasive and metastatic rates increased in relation to the extent of vascular invasion. Expressions of VEGF and p53 protein were higher and microvessel density was more prominent in tumor tissues in relation to the extent of vascular invasion. A close relation between VEGF and p53 protein expressions was also noted in tumors that showed vascular invasion. The expression of VEGF is one of the independent risk factors for vascular invasion. The postoperative outcome was poorer in patients with vascular invasion in relation to the extent of vascular invasion. CONCLUSIONS: Our findings show that gastric cancers with characteristics of vascular invasion have greater intratumoral angiogenesis and that VEGF and p53 overexpression is associated with intratumoral angiogenesis and metastases to distant organs.

Gastric cancer with high telomerase activity shows rapid development and invasiveness.

Telomerase activity was reported to be activated in most immortal cells and cancers. As the clinical significance of telomerase activity in human gastric cancer is controversial, we investigated this activity using a telomeric repeat amplification protocol. The telomerase activity was tentatively defined by strength of activity as follows: 3+, observed with 0.06 microg of protein; 2+, observed with 0.6 microg of protein; 1+, observed with 6 microg of protein; 0, not observed under these three conditions. Telomerase activity was detected in 35 of 39 (89.7%) gastric cancer specimens. Tumors with high telomerase activities (2+/3+) tended to have a deeper invasion, lymphatic and vascular invasion, lymph node metastasis, liver metastasis, and peritoneal dissemination, as compared to findings in case of low telomerase activities (-/1+). Thus, telomerase activity of gastric cancer tissue may reflect the malignant potential of the tumor and intensive postoperative care might be required for such patients.

Heparanase gene expression and metastatic potential in human gastric cancer.

BACKGROUND: Heparanase has been reported to play an important role in tumor progression and metastasis. We examined the relationship between heparanase mRNA expression and biological factors regarding invasion and metastasis of human gastric cancer. MATERIALS AND METHODS: In 63 human gastric carcinomas, 42 adjacent normal gastric tissues and four gastric cancer cell lines, heparanase mRNA expression was evaluated using reverse transcription PCR (RT-PCR). Total RNA obtained from human peripheral blood (PB) leucocyte and placenta were used as positive controls. The relationship between heparanase mRNA expression and various clinicopathological factors were analyzed. RESULTS: The heparanase mRNA expression evaluated with RT-PCR revealed that 31 out of 63 gastric cancer tissues (49%), 11 out of 42 normal gastric tissues (26%) and 4 gastric cancer cell lines were positive. The positive rate in cancer tissues was significantly higher than that in normal tissues (p<0.05). In the heparanase mRNA-positive cancer tissues, venous invasion was frequent (p<0.05) and the histological differential grade was significantly poorer than in negative cases (p<0.01). CONCLUSION: We propose that heparanase mRNA expression is involved in invasion and development of human gastric cancer and detection of this expression may be a factor related to metastasis and prognosis of such patients.

Application of quantitative RT-PCR using "TaqMan" technology to evaluate the expression of CK 18 mRNA in various cell lines.

Reverse transcriptase polymerase chain reaction (RT-PCR) is often used for sensitive detection of micrometastasis in peripheral blood, lymph nodes and bone marrow. While the utility of this method has been documented, it also has limitations in the detection of micrometastasis. The mRNA of target genes can be detected in healthy donors or in samples used for negative control, therefore the non-quantitativeness of conventional RT-PCR has been called into question. We analyzed the expression level of cytokeratin (CK) 18 mRNA in established esophageal and gastrointestinal carcinoma cell lines and non-epithelial cells, using quantitative RT-PCR, based on real time 'TaqMan TM' technology. CK 18 mRNA is more highly expressed in carcinoma cells than in non-epithelial cells. However, the expression level in non-epithelial cells was easily detected using conventional RT-PCR and agarose gel electrophoresis. In an analysis of CK 18 mRNA expression in peripheral venous blood in 13 healthy volunteers, we found that CK 18 mRNA was much less expressed than in cancer cell lines. However, the expression in all samples was at a level which was also detected using conventional RT-PCR. It would thus seem that not only qualitative, but also quantitative analysis, of the target mRNA is important to detect micrometastasis. Quantitative RT-PCR methods will make comparisons of the possible differences in expression levels of the target gene. For clinical applications, much further study is needed.

[A new microsatellite instability analysis using fluorescent primers and laser scanning].

To demonstrate the instability of microsatellite sequences, which have been linked to DNA mismatch repair deficiency and indicate a high risk of carcinogenesis, polymerase chain reaction (PCR) and electrophoretic analysis have been used. However, the electrophoretic profiles of PCR-amplified microsatellite sequences are often too complicated, and the conventional method using autoradiography has critical problems in detection characteristics and migration accuracy. First, we made use of fluorescence-labeled PCR and laser scanning to detect bands quantitatively. Second, we characterized Taq polymerase-dependent modification of the amplified microsatellite sequences and optimized the electrophoretic profiles by enzymatic modification with T4 DNA polymerase. Third, we developed a dual fluorescence co-electrophoresis system, in which both samples derived from cancer and normal tissues are electrophoresed in the same lane, in order to minimize migration errors. Furthermore, we classified all of the observed patterns of microsatellite alteration and set up new criteria for assessing microsatellite instability. Using the system developed here and the criteria we proposed, a precise judgment can be made and rates of positivity in various human malignancies may be corrected.