[Effect of peritoneum reconstruction on postoperative complications after laparoscopic low anterior resection for rectal cancer].

Objective: To determine the effect of peritoneum reconstruction on postoperative complications after laparoscopic low anterior resection (LAR) for rectal cancer. Methods: Retrospective cohort study and propensity score matching were conducted. Case inclusion criteria: (1) pathologically confirmed rectal adenocarcinoma; (2) 18 to 80 years; (3) patients with middle to low rectal cancer undergoing laparoscopic LAR; (4) patients staging cT1-4aN0-2M0 or ycT1-4aN0-2M0 after neoadjuvant therapy; (5) the distance of 4-10 cm from tumor low margin to anal verge. Exclusion criteria: (1) abdominal surgery history (except appendicitis, cholecystitis, ectopic pregnancy); (2) anastomosis above the peritoneal reflection; (3) tumor distant metastasis or clinical staging of T4b during surgery; (4) conversion to open surgery; (5) severe incapacitating disease (American Society of Anesthesiologists classification IV or V, ASA). A total of 666 patients with middle to low rectal cancer undergoing laparoscopic LAR in The First Affiliated Hospital of Army Medical University from January 2017 to June 2020 were enrolled. There were 473 males and 193 females with the median age of 59 (18-80) years. Laparoscopic LAR with peritoneum reconstruction was performed in 188 cases (PR group), and laparoscopic LAR without peritoneum reconstruction was performed in 478 cases (NPR group). After 1:1 propensity score matching according to 1:1 based on age, gender, body mass index, TNM staging, ASA classification, intraoperative blood loss, distance from tumor low margin to anal edge, 153 cases were included in each group. Postoperative complications were classified according to Clavien-Dindo classification. Anastomotic leakage was defined and graded according to the International Study Group of Rectal Cancer (ISGRC) criteria. Results: After propensity score matching, there were no significant differences in baseline demographic characteristics between the 2 groups (all P>0.05), indicating that these two groups were comparable. (1) Operative conditions: All the patients in both groups completed operation successfully. Compared with the NPR group, the PR group had longer operation time [(181.3±60.3) minutes vs. (168.9±51.5) minutes, t=2.185, P=0.029], shorter postoperative median hospital stay [8 (7, 10) days vs. 9 (7, 11) days, Z=-2.282, P=0.022], and the differences were statistically significant (P<0.05). (2) Postoperative complications: The overall morbidity of postoperative complication in PR group and NPR group was 20.3% (31/153) and 24.2% (37/153) respectively, and the incidence of anastomotic leakage was 9.8% (15/153) and 11.1%(17/153) respectively, whose differences were not statistically significant (both P>0.05). Compared with NPR group, PR group had lower morbidity of grade III to IV complications [3.9% (6/153) vs. 11.1% (17/153), χ(2)=5.688, P=0.017] and lower secondary operation rate [1.3% (2/153) vs. 5.9% (9/153), χ(2)=4.621, P=0.032], the differences were statistically significant (both P<0.05). Though PR group had lower incidence of grade C anastomoic leakage [1.3% (2/153) vs. 3.9% (6/153), χ(2)=2.054, P=0.152], but the differences were not statistically significant. (3) Postoperative inflammation: The difference of the procalcitonin level of both PR and NPR groups at postoperative 1-d, 3-d, and 5-d was statistically significant (F=5.222, P=0.010) in time-dependent manner, while the difference was not significant in the interaction effect (P>0.05). No statistically significant differences in the C-reactive protein level between two groups at postoperative 1-d, 3-d, and 5-d were found (all P>0.05). Conclusion: Peritoneum reconstruction in laparoscopic LAR can decrease the morbidity of postoperative complication of grade III to IV and the reoperation rate, and plays an important role in controlling the inflammatory reaction, which has great clinical value.

LncRNA MEG3 aggravates palmitate-induced insulin resistance by regulating miR-185-5p/Egr2 axis in hepatic cells.

OBJECTIVE: Long non-coding RNA (LncRNA) has been reported to play an important role in type 2 diabetes (T2D). We investigated the role of LncRNA maternally expressed gene 3 (MEG3) and its potential interaction with miR-185-5p in palmitate-induced hepatocyte insulin resistance. PATIENTS AND METHODS: High-fat diet (HFD) mice and insulin resistant hepatocyte were employed. Relative mRNA expressions of MEG3, miR-185-5p, and early growth response proteins-2 (Egr2) were measured by qRT-PCR. Western blot was performed to evaluate Egr2 protein expression levels. Glycogen contents and plasma insulin levels were tested by the corresponding assay. RESULTS: MEG3 and Egr2 were upregulated, but miR-185-5p was downregulated in palmitate-treated insulin resistance hepatocytes and HFD mice. MEG3 knockdown alleviated the influence of palmitate on insulin resistance in vitro and in vivo. miR-185-5p expression was upregulated upon MEG3 knockdown. Expression of Egr2 was positively correlated with MEG3 knockdown or overexpression, which could be negatively managed by abnormal expression of miR-185-5p. CONCLUSIONS: Our data demonstrated that LncRNA MEG3 aggravated palmitate-induced insulin resistance by regulating miR-185-5p/Egr2 axis, providing new insights into T2D therapeutic strategies.

MiR-638 serves as a tumor suppressor by targeting HOXA9 in glioma.

OBJECTIVE: MiR-638 is constantly downregulated and serves as a tumor suppressor in various cancers. Its role in gliomas remains unclear. This study is designed to investigate the clinical significance and the pathogenic role of miR-638 in human gliomas. PATIENTS AND METHODS: Quantitative Real-time PCR was performed to analyze the expression of miR-638 in the tumor and adjacent tissues of 24 glioma patients. The association between the expression of miR-638 and clinical features were examined. Survival of patients was studied by Kaplan-Meier curves. The impact of miR-638 on cell growth and apoptosis was determined by CCK-8 assay, colony formation assay, cell cycle analysis and Annexin V-FITC-PI apoptosis assay. The effect of miR-638 on HOXA9 was determined by luciferase assay and Western blot. The effect of miR-638 and HOXA9 on expression of oncogenes, Cyclin D1 and C-MYC was determined by Western blot. RESULTS: MiR-638 expression was constantly downregulated in glioma tumor tissue, which is negatively correlated with the WHO grade. MiR-638 expression was associated with clinical features such as tumor size, KPS score and WHO grade. Patients with low miR-638 had a worse overall survival than those with high expression. Experimentally, miR-638 directly targeted HOXA9 to suppress its expression, leading to attenuations of cell proliferation, colony formation and cell cycle progression and enhanced basal apoptosis level. MiR-638/HOXA9 axis also suppressed the expression of Wnt/beta-catenin-regulated oncogenes, Cyclin D1 and C-MYC. CONCLUSIONS: MiR-638 is a constantly downregulated microRNA in gliomas and is associated with its prognosis. MiR-638 regulates cellular malignancy of gliomas through targeting HOXA9. Thus, miR-638/HOXA9 signaling axis may have therapeutic potential in gliomas.

IMP3 promotes TNBC stem cell property through miRNA-34a regulation.

OBJECTIVE: To explore the expression and function of insulin-like growth factor II (IGFII) mRNA binding protein (IMP3) in the Triple Negative Breast Cancer (TNBC). MATERIALS AND METHODS: According to previously reported gene expression array, we found that IMP3 had significantly higher expression in the CD44+CD24-ESA+ cell cluster, tumor initiating cell or cancer stem cell (CSCs), compared to other tumor cells. Based on the GEO database (GEO accession No. GSE6883), we detected the mRNA levels of IMP 1,2 and 3 by quantitative polymerase chain reaction (q-PCR) in CD44+CD24-ESA+ cell cluster and other breast tumor cell clusters. Besides, we measured IMP3 expression in microsphere of breast cancer, which exerted more significant tumor stem cell properties. The effects of IMP3 on breast cancer cell stem cell properties were studied by RNA interference and overexpression approaches in vitro. Furthermore, we predicted and identified microRNA, which could target and regulate IMP3 from bioinformatics analysis, and verified the interaction by luciferase assays and rescue experiments. RESULTS: Previously reported data showed that IMP3 expression was significantly upregulated in CD44+CD24-ESA+ cell cluster from breast cancer tissues. Besides, we found IMP3 had higher expression in mesenchymal cells rather than epithelial cells, which was also significantly elevated in SUM159 and T49D cell lines cultured as microsphere rather than adherent cells or differentiated cells. CD44+CD24-ESA+ cell cluster proportion was significantly decreased after silencing IMP3 in SUM1315, and its ability to develop into microsphere was significantly inhibited. By re-expressing IMP3 in SUM315, we restored the self-renewal capacity and tumorigenesis potential of SUM315. Through relative predicting website, we found several miRNAs which could regulate IMP3. miR-34a with highest score was chosen for further analysis. Mimicking miR-34a significantly downregulated IMP3 expression and inhibited its ability to develop into microsphere, while overexpressing IMP3 could rescue this process. CONCLUSIONS: IMP3 plays a vital role in maintaining stem cell properties of breast cancer cells, which could be regulated by mir-34a.

[Mononuclear cells of umbilical cord blood differentiation to granulocyte cell in vitro].

Objective: To explore an optimal method for granulocyte cell production from umbilical cord blood mononuclear cells. Methods: Erythrocytes were precipitated by hydroxyethyl starch. Mononuclear cells were isolated through Ficoll density gradient centrifugation. Different media, additives and cultivation model were chosen for granulocyte induction. Cell morphology was observed by microscopy, and cell phenotype was detected by flow cytometry. The CD18 expression of granulocytes was tested by immunofluorescence assay, and phagocytosis test was executed as well. Results: Compared to fetal bovine serum (FBS) treatment group, cell viability, counts and differentiation rate of granulocytes induced by X-VIVO(TM) 15 combined with TPO, SCF, G-CSF but without FBS were superior. And X-VIVO(TM)15 medium was better than SCGM medium at effectiveness and cost. Using two-stage mode of hematopoietic stem cell expansion followed by granulocyte induction with X-VIVO(TM)15 combining TPO, SCF and G-CSF, cell proliferation was nearly 132 times at day 21. Flow cytometry showed that the differentiation was lagged in 2-stage mode than in direct induction mode, CD15 expression was (69.60± 1.06) % vs (97.73±0.39) %; Wright-Giemsa staining demonstrated mature granulocytes; immunofluorescence showed the expression of lysosomal proteins CD18. A strong phagocytic function of mature granulocytes was demonstrated by phagotrophic efficiency of (51.43±0.05) %. And granulocyte had chemotaxis ability under the role of chemotactic factor IL-8. Conclusion: Optimized culture media and cultivation mode are achieved for functional granulocytes induction in vitro.

Successful Rescue of Late-onset Antibody-mediated Rejection 12 Years After Living-donor Intestinal Transplantation: A Case Report.

BACKGROUND: Antibody-mediated rejection (ABMR) has recently surfaced as a potential form of graft dysfunction after intestinal transplantation. METHODS: We present a case of an intestinal transplant recipient who developed late-onset ABMR 12 years after living-donor transplantation. An 18-year-old male recipient with a history of extensive intestinal resection secondary to acute bowel volvulus exhibited an excellent baseline immune profile for transplantation, including ABO-identical and HLA-haploidentical to the donor; a negative cross-match with a panel reactive antibody of 3.0%. RESULTS: Post-transplantation immunosuppression consisted of tacrolimus, mycophenolate mofetil (MMF), and prednisone within the first year, followed by tacrolimus and MMF in the second year, and maintenance with tacrolimus monotherapy thereafter. The recipient experienced a single episode of indetermined acute cellular rejection 3 months after transplantation. Since then, he did not require any parenteral nutrition and had completely reintegrated with society. Twelve years later, the patient developed persistent diarrhea associated with transplant biopsy diffuse C4d deposition and circulating donor-specific antibodies. After the use of rituximab and intravenous immunoglobulin, the recipient stabilized 17 years after transplantation with complete recovery of intestinal mucosal damage. CONCLUSION: Late-onset ABMR can emerge after transplantation and must be considered a possible cause of graft dysfunction in long-term intestinal transplantation survivors.

Lipoxin A4 analog attenuates morphine antinociceptive tolerance, withdrawal-induced hyperalgesia, and glial reaction and cytokine expression in the spinal cord of rat.

Spinal neuroinflammation has been shown to play an important role in the development of morphine tolerance and morphine withdrawal-induced hyperalgesia. Lipoxins are endogenous lipoxygenase-derived eicosanoids that can function as "braking signals" in inflammation. The present study investigated the effect of 5 (S), 6 (R)-lipoxin A4 methyl ester (LXA4ME), a stable synthetic analog of lipoxin A4, on the expression of antinociceptive tolerance and withdrawal-induced hyperalgesia in chronic morphine-treated rats. Chronic morphine administration through repeated subcutaneous injection induced the development of hyperalgesia and the expression of spinal antinociceptive tolerance to morphine. However, LXA4ME treatment significantly attenuated the development of hyperalgesia and the expression of spinal antinociceptive tolerance to intrathecal morphine in both mechanical and thermal test. Moreover, the administration of LXA4ME during the induction of morphine tolerance inhibited the activation of microglia and astrocytes; reduced the expression of proinflammatory cytokines interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α); upregulated the expression of anti-inflammatory cytokines IL-10 and transforming growth factor-ß1 (TGF-ß1); and inhibited nuclear factor-kappa B (NF-κB) activation at the L5 lumbar spinal cord. These results suggest that treatment of LXA(4)ME provides a potential preventative or therapeutic approach for morphine tolerance and associated abnormal pain sensitivity.

Toughening mechanisms in iron-containing hydroxyapatite/titanium composites.

Pure hydroxyapatite (HA) is brittle and it cannot be directly used for the load-bearing biomedical applications. The purpose of this investigation was to develop a new iron-containing HA/titanium composite via pressureless sintering at a relatively low temperature with particular emphasis on identifying the underlying toughening mechanisms. The addition of iron to HA/titanium composites led to a unique and favorable core/shell microstructure of Ti-Fe particles that consisted of outer titanium and inner iron, and good interfacial bonding with HA matrix. While the relative density, hardness and Young's modulus reduced, the flexural strength, fracture toughness, fatigue resistance, and the related fracture surface roughness increased significantly with increasing amount of Ti-Fe particles. Different toughening mechanisms including crack bridging, branching and deflection were observed in the composites, thus effectively increasing the crack propagation resistance and resulting in a substantial improvement in the mechanical properties of the composites.

Glioblastoma stem cells produce vascular endothelial growth factor by activation of a G-protein coupled formylpeptide receptor FPR.

Glioma stem cells (GSCs), or stem cell-like glioma cells, isolated from malignant glioma cell lines, were capable of producing vascular endothelial growth factor (VEGF). However, the exact role of such tumour cells in angiogenesis remains unknown. In this study, we isolated a small proportion of CD133+ GSCs from the human glioblastoma cell line U87 and found that these GSCs possessed multipotent differentiation potential and released high levels of VEGF as compared with CD133(-) tumour cells. The CD133+ GSCs also formed larger xenograft tumours that contained higher VEGF immunoreactivity and denser microvessels. Moreover, GSCs expressed a functional G protein-coupled formylpeptide receptor FPR, which was activated by a chemotactic peptide ligand, N-formylmethionyl-leucyl-phenylalanine (fMLF), to mediate calcium flux and the production of VEGF by GSCs. Our results indicate that FPR expressed by human GSCs may play an important role in glioma angiogenesis.

Pain responses in patients with upper-extremity disorders.

The purpose of this study was to evaluate the relationship between pain response factors and upper-extremity disorders associated with work-related compensable disorders. In this retrospective study, the charts of 113 patients were examined. Compensation was not found to have any statistically significant association with pain levels. The degree of functional overlay in these patients, indicated by pain questionnaire scores, differed only slightly between compensated and noncompensated patients and indicated no significant difference between the 2 groups, except that the compensated group used a higher number of descriptors to describe their pain (p = .0143). These results indicate that compensation affects the verbalization of pain but does not affect the degree of pain experienced. Working status was found to be significantly correlated with a better ability to cope with stress at home, suggesting that employment status may be a more important factor than compensation status in the presentation of these patients.

A case-control study of risk factors of nasopharyngeal carcinoma.

Risk factors and nasopharyngeal carcinoma (NPC) in residents of Guangzhou city were investigated in a case-control study: 104 cases were compared with an equal number of age, sex, and neighborhood-matched controls. The results of multiple conditional regression of logistic model showed that plum vegetable (adjusted OR = 1.81, 95% CI 1.01-3.33), preserved prune (adjusted OR = 2.95, 95% CI 1.04-8.41), no separate kitchen (if >35 years, adjusted OR = 1.98, 95% CI 1.24-3.75), kitchen range without chimney (if >10 years, adjusted OR = 2.72, 95% CI 1.56-4.73) and hereditary factor (adjusted OR = 8.27, 95% CI 1.94-35.54) were significantly associated with an increased risk of NPC. Grape (adjusted OR = 0.31, 95% CI 0.17-0.58) may be a protective factor.

Effects of retinoid X receptor-selective ligands on proliferation of prostate cancer cells.

BACKGROUND: Management of prostate cancer that either is detectable by prostate specific antigen (PSA) measurements after curative intent or has spread outside of its capsule is a serious problem. Innovative, nontoxic approaches to the disease are required. One approach might be therapy with retinoids. Retinoid activities are mediated by two distinct families of transcription factors: the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which can induce transcriptional activation through specific DNA sites or by inhibiting the transcription factor AP-1 that usually mediates cellular proliferative signals. The RARs require heterodimerization with RXRs. RXRs can form either heterodimers or homodimers; and the latter can bind to DNA response elements that are distinct from those bound by the RAR/RXR heterodimers. METHODS: A series of novel synthetic retinoids that selectively interact with RXR/RXR homodimers or RAR/RXR heterodimers, or that selectively inhibit AP-1 activity without activating transcription were evaluated for their ability to inhibit clonal growth of three human prostate cancer cell lines (PC-3, DU-145, and LNCaP). RESULTS: Several notable findings were: 1) RXR-selective retinoids, such as SR11246, were able to inhibit the clonal growth of prostate cancer cells. In contrast, SR11246 had little effect on clonal growth of myeloid leukemic cells. 2) RAR-selective retinoids also inhibited clonal growth of prostate cancer cells. 3) The retinoid (SR11238) with potent anti-AP-1 activity had no effect on the clonal growth of prostate cancer cells. CONCLUSIONS: This study shows that both RXR- and RAR-selective retinoids are worthy of further study and may be candidates for future clinical trials in prostate cancer.

Effects of potent vitamin D3 analogs on clonal proliferation of human prostate cancer cell lines.

BACKGROUND: Management of prostate cancer that has spread outside of the prostate capsule is a difficult problem. Innovative, non-toxic approaches to the disease are required. New, relatively non-toxic vitamin D3 analogs have recently been synthesized. We report that several of these compounds have marked antiproliferative effects on prostate cells. METHODS: The clonal antiproliferative activity of five novel analogs of 1,25 dihydroxyvitamin D3 [1,25(OH)2D3, (cmpd C)] as well as 1,25(OH)2D3 itself was tested on three human prostate cancer cell lines (PC-3, LNCaP, and DU-145). The analogs were 20-epi-22oxa-24a,26a,27a-tri-homo-1 alpha,25(OH)2D3 (code name: KH 1060); 24a26a27a-tri-homo-22,24-diene-1 alpha,25(OH)2D3 (code name: EB 1089); 1,25(OH)2-16ene-D3 (code name: HM); 1,25(OH)2-16ene-23yne-D3 (code name: V); 1,25(OH)2-20-epi-D3 (code name: MC 1288)]. RESULTS: With the parent compound [1,25(OH)2D3], the effective dose that inhibited 50% clonogenic growth of PC-3 and LNCaP was 10(-8)M and 7 x 10(-9)M, respectively. For these prostate cancer cell lines, KH 1060 was the most potent analog by an order of 25- to 35-fold as compared to cmpd C. The second and third most potent analogs were HM and MC 1288. DU-145 was resistant to all the vitamin D3 analogs. The major side-effect of 1,25(OH)2D3 is the production of hypercalcemia. The relative inhibitory index (RII) was determined by comparing the antiproliferative activity of the analog to its ability to produce hypercalcemia in mice injected intraperitoneally every other day. The KH 1060 had the best RTI: 50- to 70-fold greater than 1,25(OH)2D3 for PC-3 and LNCaP, respectively. CONCLUSIONS: A trial of one or more of these innovative compounds should be considered for treatment of minimal residual disease of prostate cancer.

Effects of novel retinoid X receptor-selective ligands on myeloid leukemia differentiation and proliferation in vitro.

The biologic effects of retinoids such as all-trans-retinoic acid (ATRA) and 9-cis-retinoic acid on proliferation and differentiation of hematopoietic cells are mediated by binding and activating two distinct families of transcription factors: the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). The RARs require heterodimerization with RXRs; in addition, RXRs can form homodimers, which can bind to DNA response elements that are either distinct or the same as those bound by the RAR/RXR heterodimers. Therefore, the two retinoid pathways provide sequences that are specific for effective DNA binding and activation of target genes. We have developed several series of novel synthetic retinoids that selectively interact with RXR/RXR homodimers and RAR/RXR heterodimers. We show here that SR11236 and SR11246, which are RXR-selective analogs, had little ability to inhibit clonal growth and induce differentiation of leukemic cells (HL-60 cells and fresh acute myeloid leukemia cells). However, SR11249, SR11256, and LGD1069, which activated both RXR/RXR homodimers and RAR/RXR heterodimers, could inhibit clonal growth and induce differentiation of HL-60 cells as well as leukemic cells from patients, including those with acute promyelocytic leukemia (APL). This is similar to results observed with RAR/RXR-specific ligands. Interestingly, the combination of ATRA and either SR11249, SR11256, or LGD1069 showed synergistic effects in inducing differentiation of HL-60 cells. A retinoid (SR11238) with strong anti-AP-1 activity that did not activate the RARs and RXRs for gene transcription from the response element TREpal was inactive in our assay systems, suggesting that the antiproliferative effects of retinoids on leukemic cells is not mediated by inhibiting the AP-1 pathway. We conclude that the RAR/RXR pathway is more important than RXR/RXR pathway for differentiation and proliferation of acute myeloid leukemic cells, and certain retinoids or combination of retinoids with both RAR and RXR specificities may synergistically enhance the differentiation activity of ATRA, which may be relevant in several clinical situations.

Study of 45 cases of nasopharyngeal carcinoma with dermatomyositis.

The authors have conducted a study of 61 cases of nasopharyngeal carcinoma (NPC) with dermatomyositis (DM) admitted to the hospital between April 1964 and May 1989 and accounting for 0.027% (61/226, 183) of all the malignant tumors at the hospital and 0.086% (61/70,899) of the nasopharyngeal carcinoma cases, during that time period. We have analyzed 45 cases with complete data, using equal number of age-, sex-, and stage-matched cases with only NPC as control. The findings show a 5- and 10-year survival rate and distant metastatic rate of 50.4, 34.5, and 40.5% respectively, for NPC with DM, and 57.8, 55.2, and 56.5% for controls. The results indicated that the radiotherapy with prednisone treatment not only is quite effective but also will not result in a significantly increased rate of distant metastasis.

Absence of WAF1 mutations in a variety of human malignancies.

A newly cloned gene named wild-type p53-activated fragment 1 (WAF1; also known as p21, Pic-1, Cip-1, or SDI1) is directly regulated by p53 and can itself suppress tumor cell growth in culture. Induction of expression of WAF1 may be an important means by which cells with DNA injury arrest their growth to repair DNA or undergo apoptosis. Based on the hypothesis that mutations of this gene may play a role in carcinogenesis, we have studied 351 DNAs from 14 kinds of malignancies, as well as 36 human transformed cell lines, for alterations of WAF1 gene by single-strand conformation polymorphism analysis of polymerase chain reaction amplification of the DNA coding region of the WAF1 gene. No abnormal band shifts of WAF1 were noted in any of the samples or cell lines, but three major variants in exons 2 and 3 of the gene were found that are consistent with the existence of two different DNA polymorphisms. Sequence analysis of the amplified products producing these three variants in each exon from normal DNAs confirmed the presence of the polymorphisms in the WAF1 gene. Of 290 selected tumor samples previously evaluated for p53 mutations by single-strand conformation polymorphism, 90% had no detectable p53 alterations. In summary, mutations within the coding portion of the WAF1 gene were undetectable in a large series of human tumors, many of which had a normal p53 gene. This suggests that WAF1 alterations are generally caused indirectly, through p53 mutations rather than through intragenic mutation of the WAF1 itself.

Myeloid differentiation mediated through retinoic acid receptor/retinoic X receptor (RXR) not RXR/RXR pathway.

Retinoids, such as all-trans-retinoic acid and 9-cis-retinoic acid, are naturally occurring ligands of the nuclear retinoic acid receptors (RARs). In concert with binding of ligand, these receptors from heterodimers with the retinoic X receptor (RXR) and transactivate RAR/RXR-responsive genes. Retinoids can differentiate leukemic cell lines in vitro and induce clinically complete remissions in patients with acute promyelocytic leukemia. Synthetic ligands to the RAR and RXR receptors have been developed that selectively bind and activate RAR/RXR (TTAB) and RXR/RXR dimers (SR11217). We investigated the affect of these ligands, either alone or in combination, on in vitro growth and differentiation of cells from the HL-60, KG-1, THP-1, and WEHI-3 myeloid cell lines as well as on clonal growth of fresh myeloid leukemic blasts from patients. Clonal inhibition of proliferation of these cells was studied in soft agar cultures. Cells were plated in the presence of either one or a combination of retinoids at concentrations of 10(-5) to 10(-10) mol/L. TTAB inhibited 50% clonal growth at an effective dose (ED50) that was about 1,000-fold lower than the concentration of SR11217 required to achieve an ED50 for the same leukemic cells. Combination of both ligands at a variety of concentrations showed no synergistic effects. Superoxide production (nitroblue tetrazolium reduction) and CD11b expression as parameters of differentiation of HL-60 cells were also examined. Results paralleled those of clonal growth, with SR11217 being markedly less potent than TTAB. These results show that the ligand selective for RXR-homodimers has little effect on either inducing differentiation or inhibiting clonal growth of leukemic cells. The differentiating and antiproliferative effects of retinoids are mainly induced through RAR/RXR heterodimers, and development of therapeutic analogs should focus on this category of retinoids.

Transactivational and DNA binding abilities of endogenous p53 in p53 mutant cell lines.

Cells with divergent mutant alleles of the p53 gene have different biological and biochemical properties in vitro. Increasing evidence indicates that p53 is a transcriptional activator, and recently, high affinity DNA binding sites for p53 have been identified. The purpose of this study was to determine in vivo, the effect that various mutant p53 proteins have on their ability to mediate transactivation and to bind specifically to DNA. Either a p53 responsive or control reporter gene was transfected into 18 human carcinoma cell lines, having various p53 mutations, either with or without a wild-type p53 expression vector. The CAT activity and DNA gel retardation were studied to measure transactivation and DNA binding by these endogenous p53s. As expected, the endogenously produced wild-type p53 binds to DNA binding sequences and can transactivate a reporter construct containing a p53 high affinity DNA binding site. Four of five cell lines with homozygous p53 mutations at codon 273 (273His), contained p53 which had the ability to bind to p53 DNA binding sequences and transactivate. In contrast, all the homozygous, non-codon 273 mutant p53s (156Pro, 175His, 223Leu, 248Gln, 248Trp, 280Lys) present in the other cell lines had no transactivating ability. These findings suggest that the biology of cancers with mutations at codon 273 may be different than those with p53 mutations at other sites. The p53 from WRO, a thyroid carcinoma cell line with p53 mutation at codon 223 (223Leu), was able to bind p53 DNA recognition sequences, but was unable to transactivate. Interestingly, in a vulvar carcinoma cell line (A431) with a p53 mutation at codon 273 (273His), the p53 was unable to transactivate and gave an aberrant band on gel retardation. Both CEM and SK-UT-1, which have compound heterozygous mutations at codons 175/248 (175His/248His), produced p53 which can complex with DNA, as well as transactivate. In contrast, the p53 in cell lines with either homozygous 175His or 248His p53 mutations, were unable either to transactivate or bind to the p53 response element. A cell line (NPA) heterozygous for 266Glu p53 mutation, was able to efficiently transactivate a reporter containing a p53 DNA binding site, therefore showing no evidence of a dominant negative effect of the endogenous p53 mutant allele. In summary, this in vivo study further supports the idea that different p53 mutant alleles have various properties which may affect their function.