[Efficacy of different regimens and prognostic factors in patients with first relapsed multiple myeloma treated after front-line bortezomib, cyclophosphamide, and dexamethasone].

Objective: To analyze the efficacy of second-line regimens and prognostic factors in patients with first-relapsed multiple myeloma (MM) treated with bortezomib, cyclophosphamide, and dexamethasone (BCD). Methods: A retrospective cohort study. Clinical data were collected in first-relapsed MM patients after BCD treatment from three tertiary hospitals in north China from July 2009 to October 2022. Patients were classified according to the second-line regimen into the immunotherapy group, single novel agent group [either proteasome inhibitor (PI) or immunomodulatory drug (IMiD)], combination treatment group (both PI+IMiD), and traditional treatment group. Responses to second-line regimens and survival data were analyzed. The Kaplan-Meier method was used for survival analysis and the Cox proportional risk model was used for univariate and multivariate analyses. Results: A total of 217 patients were enrolled including 8.8% (19/217) in the immunotherapy group, 48.4% (105/217) in the PI/IMiD group, 29.9% (65/217) in the PI+IMiD group, and 12.9% (28/217) in the traditional treatment group. The median age was 62 years (range 31-83 years) and 56.2% (122/217) were males. The overall response rates (ORRs) in the four groups were 94.7% (18/19) vs. 56.2% (59/105) vs. 73.8% (48/65) vs. 32.1% (9/28) (χ2=24.55; P<0.001), respectively. The progression-free survival (PFS) of the second-line regimens (2ndPFS) was 17.7 vs. 9.0 vs. 9.2 vs. 4.6 months (χ2=22.74; P<0.001), respectively, among which patients in the PI/IMiD and PI+IMiD groups had comparable 2ndPFS (χ2=1.76; P=0.923). Patients with high-risk cytogenetic abnormalities (HRCAs) achieved the longest 2ndPFS of 22.0 months in the immunotherapy group (χ2=15.03; P=0.002). Multivariate analysis suggested that immunotherapy (HR=0.11, 95%CI 0.05-0.27), achievement of efficacy of partial response or better (HR=0.47, 95%CI 0.34-0.66), and non-aggressive relapse (HR=0.25, 95%CI 0.17-0.37) were independent prognostic factors of 2ndPFS. Conclusion: In this real-world study, immunotherapy was associated with a more favorable efficacy and PFS for first-relapsed MM patients after BCD treatment, with similar outcomes in patients with HRCAs.

[Correlation between urinary sediment spectra and pathological patterns of renal biopsies].

OBJECTIVE: To investigate the clinical value of urinary sediment analysis, a non-invasive diagnostic means, in the evaluation of pathological patterns of renal diseases. METHODS: A total of 1 140 pairs of matched renal biopsies and fresh fasting morning urine specimens were collected from hospitalized patients in Peking University First Hospital. Their urinary sediments were examined with phase-contrast microscopy; the 24 h urine proteins were measured. Based on urinary sediment features combined with urine protein amount, the spectra of the urine sediments were classified into four types, Type I: hematuria-dominant, with multiple cells and casts; Type II: proteinuria-dominant, with hyaline or fine-granular casts but scanty cells; Type III: renal tubular epithelial cell(RTEC)-dominant, minor proteinuria; Type IV: non-specificurine sediments,minor proteinuria. According to the pathological lesions detected in renal biopsies, the renal diseases were classified into three patterns: proliferative glomerulopathy(P-GP), non-proliferative glomerulopathy (NP-GP) and tubulointerstitial nephropathy(TIN). The urinary sediment spectra of different pathological patterns and the correlation between urinary sediment types and pathological patterns were analyzed. Statistical analyses were performed using kappa test, and χ(2) test, and significance was accepted at P<0.05. RESULTS: (1) Of the 840 cases of matched urine samples and renal biopsies, 419 cases were diagnosed with P-GP; 375 cases with NP-GP; 46 cases with TIN respectively. (2) The spectra of urine sediments were associated with pathological patterns of the renal biopsies, and 84.0% of the patients with P-GP manifested type I urine sediments; 93.1% of the patients with NP-GP had type II urine sediments; 67.4% of the patients with TIN had type III urine sediments. (3) The correlation between the urinary sediment types and renal pathological patterns was validated in an additional 300 matched samples. The positive predictive values of urinary sediment spectra in predicting renal pathological lesions were 84.8% for typeI to P-GP, 86.0% for type II to NP-GP and 73.7% for type III to TIN, respectively. CONCLUSION: As a non-invasive diagnostic means, the urinary sediment analysis is valuable in the evaluation of pathological patterns of renal diseases.

Human and mouse IFN-beta gene therapy exhibits different anti-tumor mechanisms in mouse models.

Previously, we suggested that local human interferon-beta (IFN-beta) gene therapy with replication-defective adenoviral vectors can be an effective cancer treatment. Clinical trials to treat cancers with adenovirus expressing the human IFN-beta gene (IFNB1) has been planned. As a continued effort to explore the mechanisms of action of human IFN-beta gene therapy that can occur in the clinical setting, we tested mouse IFN-beta gene therapy in human xenograft tumors in both ex vivo and in vivo models. Delivery of the mouse IFN-beta gene (Ifnb) caused tumor inhibition; this effect was dependent on the indirect anti-tumor activities of IFN-beta, notably a stimulation of natural killer cells. IFN-beta does not show cross-species activity in its anti-proliferative effect and mouse IFN-beta does not cause as significant an anti-proliferative effect on mouse tumor cells as human IFN-beta causes on human tumor cells. Therefore, we believe that mouse models using either human IFN-beta or mouse IFN-beta gene transfer do not capture all aspects of the action of adenovirus-mediated human IFN-beta gene therapy that may be present in the clinical setting. Due to its multiple mechanisms of action, human IFN-beta gene therapy may be effective in treating human cancers that are either sensitive or resistant to the direct anti-proliferative effect of IFN-beta.

[Integrin-ligands binding reaction upregulates the antioxidant activity of rabbit bronchial epithelial cells].

Antioxidant activity of bronchial epithelial cells (BECs) plays an essential role in preventing the airway epithelium integrity from damage in structure and function. Integrin expressed by BECs is the receptor of extracellular matrix such as fibronectin (Fn), and it is involved in modulation of proliferation, differentiation and metabolism of the cells. In order to test the hypothesis that integrin-ligand binding reaction supports the ability of cells to withstand oxidant attack, the present study evaluated the antioxidant activity of primary cultured rabbit BECs treated with fibronectin or its sequence Arg-Gly-Asp (RGD peptide), by determining changes in the activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) and in the level of glutathione (GSH). The results are as follows: (1) Fn (10 micrograms/ml) increased significantly the activity unit of GSH-Px (P < 0.05, n = 5), which was inhibited by calmodulin-inhibitor W7 (10(-5) mol/L) (P < 0.05). Both Fn (5-20 micrograms/ml) and RGD (15-60 micrograms/ml) showed a dose-dependent upregulatory effect (respectively r = 0.93 and r = 0.73). (2) Treatment with Fn increased SOD activity (P < 0.01, n = 7), which was abolished by W7 (P < 0.01). (3) Catalase activity was also stimulated by Fn (P < 0.05, n = 6) and reversed by W7 (P < 0.01). (4) A dose-dependent increase of GSH level was observed in both Fn (r = 0.82) and RGD treatment (r = 0.84). The data suggest that the binding of integrin with extracellular matrix can upregulate activity of antioxidant enzymes, and increase the content of GSH and improve the ability of BECs to resist oxidant injury.

[Resisting injury and protective effect of fibronectin on bronchial epithelial cells].

AIM AND METHODS: To test the hypothesis that activation of integrin molecules play a role on protecting bronchial epithelial cells (BEC) from oxidant injury. Based on a cell-injury model made by ozone (1.5 x 10(-6)) exposure, the present study investigated the protective effect of fibronectin (Fn), a kind of ligand of integrin, and it's specific sequence Arg-Gly-Asp (RGD peptide) which is a recognizable domain for integrin on BEC. 3H release, LDH and MDA were measured as indexes for damage. RESULTS: (1) Ozone exposure induced a significant increase of 3H release and LDH release from BEC and elevation of MDA. (2) Pretreatment with Fn or RGD before ozone exposure markedly attenuated the release of 3H, LDH and MDA, and the effects of Fn were reversed by W7, a calmodulin inhibitor. (3) Either Fn or RGD up-regulated activity of catalase in BEC and promoted GSH synthesis in BEC. CONCLUSION: These suggests that the binding of integrin with Fn improve the antioxidant ability of BEC and lighten the damage induced by oxidant attack. The protective mechanism of Fn-integrin binding may be related with calmodulin.

[Fibronectin or RGD peptide promotes nitric oxide synthesis of rabbit bronchial epithelial cells].

To find out whether the extracellular matrix component fibronectin plays a regulatory and protective role in bronchial epithelial cells, the present study was undertaken to detect the NO released from primary cultured rabbit bronchial epithelial cells (BEC) and the activity of NO synthesase (NOS) in cells. Stress with ozone was taken as the positive control, and the effects of fibronectin (Fn) or its specific sequence Arg-Gly-Asp (RGD peptide) was observed. The results showed: (1) Ozone stress enhanced NO release from BEC, and treatment with Fn also promoted the NO production. The effect of Fn could be blocked by calmodulin inhibitor W(7). (2) Both Fn and RGD showed a dose-dependent promotion on NO release. (3) The NOS activity was significantly elevated in Fn treated group and the effect of Fn was abolished by W(7). A dose-response relation in NOS activity was observed in either Fn treated or RGD treated group. It is concluded that the binding of fibronectin or its specific sequence RGD peptide with integrins of BEC plays a role in upregulating the NOS activity and the NO release, and calmodulin may take part in the pathway of signal transduction.

[Vasoactive intestinal peptide and epidermal growth factor upregulates bcl-2 gene expression in bronchial epithelial cells].

Expression of protooncogenes bcl-2 and c-myc in cultured rabbit bronchial epithelial cell (BEC) was investigated in order to shed some light on genetic mechanisms underlying the protective antioxidant effect of pulmonary regulatory peptides, vasoactive intestinal peptide (VIP) and epidermal growth factor (EGF). Effects of these peptides and heat stress (HS) on expression of these genes were also studied. Total RNA was extracted from BEC. Bcl-2 mRNA and c-myc mRNA were cloned with the method of RT-PCR. GAPDH mRNA was used as internal control. The products of RT-PCR were separated with electrophoresis in 2% agarose gels. A computer image treating system (Stratagene eagleeye II) was used to identify the specific band and evaluate the density. The product bands of target genes bcl-2 were checked with Southern blot and oligoneucleotides probe hybridyzation. The results show: (1) a low level of bcl-2 and c-myc gene transcription occur in BEC at the resting state; (2) both VIP and EGF could promote bcl-2 and c-myc transcription, but no significant change could be found in the HS group; (3) there was a close correlation between bcl-2 and c-myc transcription (r = 0.98. P < 0.01). The above results indicate that VIP and EGF can improve the antioxidant effect of BEC by upregulating bcl-2 gene expression potently modulated by c-myc protein.

[Role of c-fos gene in the pulmonary surfactant synthesis of cultured alveolar type II cells induced by endothelin-1].

The effects of endothelin-1 (ET-1) on pulmonary surfactant (PS) synthesis of cultured alveolar type II cells (AT II) were observed. The role of c-fos gene in cellular signal transduction of ET-1 was studied by antisense technology. The results showed that: (1) ET-1 enhanced [3H] choline incorporation into AT II cells in a dose-dependent manner. (2) Protein kinase (PKC) activator PMA increased [3H] choline incorporation into AT II cells, while PKC inhibitor H7 inhibited the stimulating effect of ET-1. (3) Both ET-1 and PMA could increase the level of c-Fos protein, and H7 and c-fos antisense oligonucleotides (AS ODN) could inhibit the effects induced by ET-1 on Fos protein expression and [3H] choline incorporation. (4) The release of lactic dehydrogenase (LDH) was not different among control, ET-1, antisense oligonucleotides and sense oligonucleotides groups. The above results demonstrated that ET-1 can enhance PS synthesis of AT II cells and ET-1 stimulating the expression of c-fos gene mediated by PKC is a major signal transduction pathway of modulating PS synthesis.

[Protection from oxidant injury on airway epithelial cells and the local microenvironment modulation].

An cellular injury model of primary cultured rabbit bronchial epithelial cells (BEC) exposed to ozone was established in this study and cytoprotective effects of factors in local microenvironment of airway such as vasoactive intestinal peptide (VIP), epidermal growth factor (EGF) and heat stress were observed. These factors could lighten damage in cell and elevate the level of glutashione(GSH), which depended upon phosphorylation modulation by protein kinases and gene transcription. A low level of bcl-2 gene expression could be detected in BEC at basic state, and either VIP or EGF stimulated the transcription of bcl-2, which improved the capability of BEC to resist oxidant injury. Otherwise, EGF and heat stress increased VIP autocrine from BEC and upregulated the expression of VIP receptor on BEC, so that the protective effect of VIP can be amplified in local sites. This study confirmed that there is an anti-injury protection on airway epithelium and the protection can be adaptively modulated by the regulatory peptides in local microenvironment and exogenous stimulus.

Interferon-beta gene therapy inhibits tumor formation and causes regression of established tumors in immune-deficient mice.

Despite the potential of type 1 interferons (IFNs) for the treatment of cancer, clinical experience with IFN protein therapy of solid tumors has been disappointing. IFN-beta has potent antiproliferative activity against most human tumor cells in vitro in addition to its known immunomodulatory activities. The antiproliferative effect, however, relies on IFN-beta concentrations that cannot be achieved by parenteral protein administration because of rapid protein clearance and systemic toxicities. We demonstrate here that ex vivo IFN-beta gene transduction by a replication-defective adenovirus in as few as 1% of implanted cells blocked tumor formation. Direct in vivo IFN-beta gene delivery into established tumors generated high local concentrations of IFN-beta, inhibited tumor growth, and in many cases caused complete tumor regression. Because the mice were immune-deficient, it is likely that the anti-tumor effect was primarily through direct inhibition of tumor cell proliferation and survival. Based on these studies, we argue that local IFN-beta gene therapy with replication-defective adenoviral vectors might be an effective treatment for some solid tumors.

Interferon-beta induces S phase accumulation selectively in human transformed cells.

Interferons (IFNs) generally have been characterized as antiproliferative cytokines. The cell cycle arrest in G1/G0 phase induced by type I IFNs, especially IFN-alpha, was recognized as a manifestation of their antiproliferative effects. In this article, we report that the cell cycle block in G1/G0 is observed mainly in certain cell types, such as Daudi Burkitt's lymphoma cells. In a variety of human transformed cells, but not nontransformed primary cells, IFN-beta and IFN-alpha induced a significant increase in the S phase population. The increase appeared to be due to a continued S phase entry and subsequently a failure of S phase cells to transit efficiently into G2 and M phases. The ability of tumor cells to exhibit the S phase effect correlated with proper IFN signaling and loss or inactivation of the normal G1 checkpoint conferred by the retinoblastoma protein (pRB). Overriding the G1 checkpoint switched human nontransformed primary cells from nonresponsive to sensitive to the IFN-induced effect. Therefore, the cell cycle regulatory machinery could function, at least in part, as a determining factor that affects the IFN-induced cell cycle effect. The IFN effect in transformed cells may suggest intriguing prospects for combinatorial therapies for cancer.

Differential cell cycle effects induced by E2F1 mutants.

Expression of two types of transactivation-defective E2F1 mutants in human Rb-/- tumor cells led to an increase in the proportion of cells in the G1 phase of the cell cycle as determined by FACS analysis. Experiments revealed two different mechanisms of action. One mutant type induced a G1 arrest after the restriction point, with cells phenotypically at a cell cycle stage later than G1. The action of this mutant was, at least in part, dependent on specific DNA binding and was over-ridden by co-expression of its wild-type counterpart. The other mutant type, which is defective in DNA binding, slowed the G1 progression and restored a checkpoint for cell cycle withdrawal. The G1 phase withdrawal of these tumor cells allowed the initiation of skeletal muscle cell differentiation. Thus, E2F1 appears to have two different functions before and after the cell cycle restriction point. This report also may provide a basis for a gene therapy approach for certain human cancers.

[Cytoprotective effect of epidermal growth factor on cultured rabbit airway epithelial cells exposed to ozone].

In this study, it was observed that ozone (O3) exposure has cytotoxic effects on cultured airway epithelial cells, which was positively related with exposure duration. Both the production of malondialdehyde (MDA) and the 3H release in the exposure group were much higher than the control (P < 0.01), suggesting that lipoperoxidation occurring in the cell membrane was responsible for the cellular injury observed under O3 attack. EGF at low concentration (5 ng/ml) showed cytoprotective effect on airway epithelial cells exposed to O3 as shown by attenuated MDA production and decrease of 3H release and cytotoxic index (P < 0.01). Pretreatment with EGF could reverse the depletion of intracellular GSH content as a result of O3 exposure and increase the total glutathionein the cells. The above results suggest that the cytoprotection of EGF on airway epithelial cells may be related to its promotive effect on GSH synthesis.

The transcription factor E2F-1 is a downstream target of RB action.

Reintroduction of RB into SAOS2 (RB-/-) cells causes a G1 arrest and characteristic cellular swelling. Coexpression of the cellular transcription factor E2F-1 could overcome these effects. The ability of E2F-1 to bind to RB was neither necessary nor sufficient for this effect, and S-phase entry was not accompanied by RB hyperphosphorylation under these conditions. Furthermore, E2F-1 could overcome the actions of a nonphosphorylatable but otherwise intact RB mutant. These data, together with the fact that RB binds to E2F-1 in vivo, suggest that E2F-1 is a downstream target of RB action. Mutational analysis showed that the ability of E2F-1 to bind to DNA was necessary and sufficient to block the formation of large cells by RB, whereas the ability to induce S-phase entry required a functional transactivation domain as well. Thus, the induction of a G1 arrest and the formation of large cells by RB in these cells can be genetically dissociated. Furthermore, the ability of the E2F-1 DNA-binding domain alone to block one manifestation of RB action is consistent with the notion that RB-E2F complexes actively repress transcription upon binding to certain E2F-responsive promoters. In keeping with this view, we show here that coproduction of an E2F1 mutant capable of binding to DNA, yet unable to transactivate, is sufficient to block RB-mediated transcriptional repression.

Deregulated transcription factor E2F-1 expression leads to S-phase entry and p53-mediated apoptosis.

E2F-1 is a transcription factor suspected of activating genes required for S phase and a known target for the action of RB, the retinoblastoma gene product. Its induction in quiescent fibroblasts led to S-phase entry followed by apoptosis. E2F-1-mediated apoptosis was suppressed by coexpression of wild-type RB or a transdominant negative mutant species of p53. In contrast, coexpression of a naturally occurring loss-of-function RB mutant or wild-type p53 did not suppress the induction of apoptosis under these conditions. Thus, deregulated E2F-1 activity gives rise to proliferative and apoptotic signals. p53 appears to participate in the execution of the latter.

Probing the role of histidine-372 in zinc binding and the catalytic mechanism of Escherichia coli alkaline phosphatase by site-specific mutagenesis.

In the X-ray structure of Escherichia coli alkaline phosphatase at 2.0-A resolution, His-372 was found only 3.8 A away from the zinc and forms a hydrogen-bonding interaction with Asp-327, a bidentate ligand of the zinc at the M1 site. However, His-372 does not directly interact with the zinc atom at the M1 site. In order to investigate the role of the side chain of His-372 in zinc binding and the catalytic mechanism of Escherichia coli alkaline phosphatase, site-directed mutagenesis was used to convert His-372 to alanine. The fact that the His-372-->Ala enzyme has similar zinc binding affinity as the wild-type enzyme indicates that His-372 is not involved in zinc binding at the M1 site. However, the altered kinetic behavior of the mutant enzyme compared to the wild-type enzyme suggests that the imidazole ring of His-372 plays an indirect role in the catalytic mechanism of the enzyme. The hydrolysis activity of the His-372-->Ala enzyme at pH 8.0 is 10-fold lower than that of the wild-type enzyme. In the presence of a phosphate acceptor at pH 8.0, the mutant enzyme is approximately 80% as active as the wild-type enzyme. Therefore, the His-372-->Ala mutation selectively enhances the transphosphorylation activity of the enzyme. The His-372-->Ala enzyme also exhibits 4- and 30-fold decreases in Km as compared to the wild-type enzyme in 0.1 M MOPS buffer and 1.0 M Tris, buffer at pH 8.0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a growth suppression domain within the retinoblastoma gene product.

To date, all naturally occurring retinoblastoma susceptibility gene (RB) mutations known to be compatible with stable protein expression map to the T/E1A and cellular protein-binding region (the "pocket" domain). This domain extends from residue 379 to 792. When full-length RB and certain truncated forms were synthesized in human RB -/- cells, we found that the minimal region necessary for overt growth suppression extended from residue 379 to 928. A functional pocket domain and sequences extending from the carboxy-terminal boundary of the pocket to the carboxyl terminus of the protein were both necessary for growth suppression. Both sets of sequences were also required for E2F binding; hence, the two functions may be linked.

A link between double-strand break-related repair and V(D)J recombination: the scid mutation.

We show here that mammalian site-specific recombination and DNA-repair pathways share a common factor. The effects of DNA-damaging agents on cell lines derived from mice homozygous for the scid (severe combined immune deficiency) mutation were studied. Surprisingly, all scid cell lines exhibited a profound hypersensitivity to DNA-damaging agents that caused double-strand breaks (x-irradiation and bleomycin) but not to other chemicals that caused single-strand breaks or cross-links. Neutral filter elution assays demonstrated that the x-irradiation hypersensitivity could be correlated with a deficiency in repairing double-strand breaks. These data suggest that the scid gene product is involved in two pathways: DNA repair of random double-strand breaks and the site-specific and lymphoid-restricted variable-(diversity)-joining [V(D)J] DNA rearrangement process. We propose that the scid gene product performs a similar function in both pathways and may be a ubiquitous protein.