Targeting cancer metabolic pathways for improving chemotherapy and immunotherapy.

Recent discoveries in cancer metabolism have revealed promising metabolic targets to modulate cancer progression, drug response, and anti-cancer immunity. Combination therapy, consisting of metabolic inhibitors and chemotherapeutic or immunotherapeutic agents, offers new opportunities for improved cancer therapy. However, it also presents challenges due to the complexity of cancer metabolic pathways and the metabolic interactions between tumor cells and immune cells. Many studies have been published demonstrating potential synergy between novel inhibitors of metabolism and chemo/immunotherapy, yet our understanding of the underlying mechanisms remains limited. Here, we review the current strategies of altering the metabolic pathways of cancer to improve the anti-cancer effects of chemo/immunotherapy. We also note the need to differentiate the effect of metabolic inhibition on cancer cells and immune cells and highlight nanotechnology as an emerging solution. Improving our understanding of the complexity of the metabolic pathways in different cell populations and the anti-cancer effects of chemo/immunotherapy will aid in the discovery of novel strategies that effectively restrict cancer growth and augment the anti-cancer effects of chemo/immunotherapy.

Anal incontinence and bowel dysfunction after sacrocolpopexy for vaginal vault prolapse.

INTRODUCTION: This study aimed to determine the prevalence of bowel dysfunction and anal incontinence in relation to vaginal vault prolapse surgery in women hysterectomized on benign indications. METHODS: This is a case-control study where women having had sacrocolpopexy (n = 78) were compared with hysterectomized women without sacrocolpopexy (n = 233) using a bowel function questionnaire and the Cleveland Clinic Incontinence Score (CCIS). RESULTS: Sacrocolpopexy was performed on average 13.7 years (+/-11.1 SD) after the hysterectomy. Sacrocolpopexy was associated with an increased prevalence of rectal emptying difficulties (p = 0.04), incomplete rectal evacuation (p < 0.001), digitally assisted rectal emptying (p < 0.001), and use of enemas (p = 0.001). There was no overall significant difference in mean CCIS when comparing women having had vaginal vault prolapse surgery (CCIS = 2.78 +/- 4.1 SD) with those without (CCIS = 2.1 +/- 3.3 SD, p = 0.1) CONCLUSIONS: Abdominal sacrocolpopexy is associated with obstructed defecation but not anal incontinence when compared to hysterectomized controls without vaginal vault prolapse surgery.

Microsatellite variability reveals the necessity for genetic input from wild giant pandas (Ailuropoda melanoleuca) into the captive population.

Recent success in breeding giant pandas in captivity has encouraged panda conservationists to believe that the ex situ population is ready to serve as a source for supporting the wild population. In this study, we used 11 microsatellite DNA markers to assess the amount and distribution of genetic variability present in the two largest captive populations (Chengdu Research Base of Giant Panda Breeding, Sichuan Province and the China Research and Conservation Center for the Giant Panda at Wolong, Sichuan Province). The data were compared with those samples from wild pandas living in two key giant panda nature reserves (Baoxing Nature Reserve and Wanglang Nature Reserve). The results show that the captive populations have retained lower levels of allelic diversity and heterozygosity compared to isolated wild populations. However, low inbreeding coefficients indicate that captive populations are under careful genetic management. Excessive heterozygosity suggests that the two captive populations have experienced a genetic bottleneck, presumably caused by founder effects. Moreover, evidence of increased genetic divergence demonstrates restricted breeding options within facilities. Based on these results, we conclude that the genetic diversity in the captive populations is not optimal. Introduction of genetic materials from wild pandas and improved exchange of genetic materials among institutions will be necessary for the captive pandas to be representative of the wild populations.

Histological inflammatory response to transvaginal polypropylene mesh for pelvic reconstructive surgery.

PURPOSE: We prospectively evaluated the histological inflammatory response to the large polypropylene transvaginal mesh used for pelvic organ prolapse surgery. MATERIALS AND METHODS: Ten patients and 8 controls underwent vaginal punch biopsy sampling before surgery and patients also underwent it 1 year after pelvic reconstructive surgery using polypropylene mesh. Foreign body response to the mesh was assessed using a combination of histological, semiquantitative and computerized image based analysis. RESULTS: Compared to preoperative histology there was a significant postoperative increase in macrophage and mast cell counts (p = 0.03 and 0.01) but no significant changes in the count of cells involved primarily in the infectious cell response or collagen density and the elastin area fraction at the mesh-tissue interface (p = 0.2 and 0.3, respectively). Three cases of mild granuloma formation and 2 of mild erosion were observed. There was no significant change in epithelial thickness when comparing preoperative and postoperative samples. CONCLUSIONS: When used for pelvic reconstructive surgery, macroporous monofilament polypropylene mesh induces a mild but persistent foreign body reaction.

Cloning, characterization and tissue specific expression of Amur tiger (Panthera tigris altaica) IGF-I.

Insulin-like growth factor I (IGF-I) plays an important role in regulating gonad function, which is essential for normal reproduction in animals, especially in sexual receptivity and reproductive behavior. In this study, a cDNA encoding Amur tiger (Panthera tigris altaica) IGF-I was isolated from liver total RNA using RT-PCR. The IGF-I cDNA of Amur tiger (ATIGF-I) was highly homologous to that of other animals, 84.8% to rat, 93.7% to human and horse. Alignment analysis showed that the cysteine residues and many amino acid residues of putative mature ATIGF-I are highly conserved in mammalian species, confirming the high sequence homology observed in other species. DNA encoding the mature ATIGF-I peptide was ligated with pET-DsbA expression vector and highly expressed in Escherichia coli BL21 with IPTG induction. The recombinant proteins expressed existed mostly in the soluble protein fraction, and were purified with metal affinity resins. Western blotting confirmed that the recombinant proteins reacted with antibodies against IGF-I. The results obtained here should be useful for large-scale production of biological active ATIGF-I protein, as well as for further research on growth, development, and reproduction in the Amur tiger. Tissue specific expression of ATIGF-I mRNA in the Amur tiger was examined by reverse transcription-polymerase chain reaction (RT-PCR), The major ATIGF-I mRNA expression tissue was the liver, while medium signals were found in the uterus, ovary, and pituitary, and minor signals were detected in various tissues including the heart, spleen, pancreas, and kidney. The results indicate that IGF-I might play an important role in the reproductive system and in cub development in the Amur tiger.

[Enrichment of giant panda microsatellite markers using dynal magnet beads].

The 400 -600 bp DNA fractions of giant panda containing STR sequences were captured by hybridization with the oligonucleotide probes attached to streptavadin coated magnetic beads (Dynal). The enriched DNA were ligated into pGEM-T and then transformed into E. coil JM109 competent cells. In total 260 positive clones were identified from 2 880 transformants in the libraries which were screened by gamma-32 P radiolabelled probes. Finally, we got 54 sequences and successfully designed 37 pairs of STR primers for giant panda. The results showed that this method is very efficient to isolate microsatellite markers.

hTERT gene copy number is not associated with hTERT RNA expression or telomerase activity in colorectal cancer.

In a majority of malignant human tumors telomerase activity can be detected, suggesting an immortal phenotype. Expression of the reverse transcriptase subunit, hTERT, in the human telomerase complex is required for telomerase activity. The regulation of hTERT, from gene level to a fully functional protein, is still a poorly understood process. Increased copy number of the hTERT gene has been demonstrated in a significant portion of established cell lines and tumors of different origin but its relevance for telomerase activity levels is unclear. In the present study, we examined the hTERT gene copy number using fluorescence in situ hybridization (FISH) in samples from 64 colorectal carcinomas and an increased copy number (> or = 3 hTERT gene copies/nucleus) was observed in 31 cases (48%). No statistical association existed between hTERT gene copy number and hTERT RNA expression or telomerase activity. However, a significant relationship was found between an increase in hTERT gene copy number and p53 protein accumulation (p = 0.002) and aneuploidy (p = 0.036). Only 4 tumors showed microsatellite instability, 3 of which had a normal hTERT gene copy number. The data indicated that the increased copy number of the hTERT gene in colorectal carcinoma was a result of genomic instability with no obvious consequence for telomerase activity levels.

[Cloning and expression of pituitary prolactin gene in Ailuropoda melanoleuca].

The giant panda (Ailuropoda melanoleuca) is an endangered species and indigenous to China. It has been proposed that it has a highly specialized reproductive pattern with low fecundity, but little is known about its basic reproductive biology at molecular level. In this study,the pituitary prolactin (PRL) cDNA of giant panda was amplified by RT-PCR from pituitary total RNA and then cloned, sequenced and submitted to GenBank (GenBank accession No. AY161285). The sequence analysis revealed that the giant panda prolactin cDNA contains a 687-nucleotide open reading frame encoding the prolactin prohormone of 229 amino acid residues. The signal peptide contains 30 amino acid residues and the mature prolactin is composed of 199 amino acid residues. Then the DNA fragment amplified was subcloned into pGEX-4T-1 procaryotic expression plasmid and protein expression was induced by IPTG in Escherichia coil BL21. SDS-PAGE analysis revealed the PRL protein is infusible. The multiple sequence alignments revealed that the homology of giant panda is 95% to cat and pig, 80% - 70% to human, cow and goat, 52% to rat and 45.9% to mouse at the amino acid level. The 64th amino acid of giant panda prolactin is hydrophilic serine instead of hydrophobic proline of cat, goat, and cow or hydrophobic alanine of human.

Telomere attrition predominantly occurs in precursor lesions during in vivo carcinogenic process of the uterine cervix.

Although human papillomavirus (HPV) has been defined as the pathogen for cervical carcinomas, molecular events underlying the oncogenic process are unclear. As telomere dysfunction-mediated chromosomal instability and telomerase activation have been suggested as key events in carcinogenesis, we dissected the dynamic changes in telomere length, checkpoint response, and temporal profile of telomerase expression during the evolution from precursor lesions (cervical intraepithelial neoplasia, CINs) to invasive cancers of the uterine cervix in sequential samples from 16 patients. Telomeres were significantly shortened in all CIN samples and no further substantial attritions occurred in most cases with the acquisition of malignant phenotype. Very short telomeres were coupled with constitutive activation of the DNA damage response pathway (Chk2 phosphorylation) and increased cellular proliferation in those cervical specimens. Telomerase reverse transcriptase (hTERT) expression was preferably induced at advanced CINs or invasive cancers. The present finding demonstrates that excessive telomere shortening predominantly occurs in the early carcinogenesis of the uterine cervix largely prior to telomerase activation. Widespread over-erosion of telomeres or telomere dysfunction in very early stages of cervical tumorigenesis might fuel transformation processes by driving chromosomal instability.

Cloning and sequence analysis of FSH and LH in the giant panda (Ailuropoda melanoleuca).

The giant panda (Ailuropoda melanoleuca) is an endangered species and indigenous to China. It has been proposed that it has a highly specialized reproductive pattern with low fecundity, but little is known about its basic reproductive biology at the molecular level. In this report the genes encoding gonadotropin subunits alpha, follicle-stimulating hormone (FSH) beta and luteinizing hormone (LH) beta of the giant panda were amplified for the first time by RT-PCR from pituitary total RNA, and were cloned, sequenced and analyzed. The results revealed that the open reading region (ORF) of gonadotropin subunits alpha, FSH beta and LH beta are 363, 390 and 426 bp long, respectively. They displayed a reasonably high degree (74-94, 85-93, 75-91%, for alpha, FSH beta and LH beta subunits, respectively) of identity when deduced amino acids were compared with homologous sequences from partial available mammals including human, cattle, sheep, pig, rat, mouse. Three distinct differences were found at the site of 59 aa of the alpha subunit and 55 aa, 68 aa of FSH beta subunit. Our results provide an insight into understanding the mechanism of reproduction regulation and genetic characteristics of giant panda which will make an actual contribution to its conservation. In addition they lay a foundation for a further study towards producing recombinant panda FSH and LH which can be used in artificial breeding aimed to increase its captive reproductive efficiency.

Deletion of the telomerase reverse transcriptase gene and haploinsufficiency of telomere maintenance in Cri du chat syndrome.

Cri du chat syndrome (CdCS) results from loss of the distal portion of chromosome 5p, where the telomerase reverse transcriptase (hTERT) gene is localized (5p15.33). hTERT is the rate-limiting component for telomerase activity that is essential for telomere-length maintenance and sustained cell proliferation. Here, we show that a concomitant deletion of the hTERT allele occurs in all 10 patients with CdCS whom we examined. Induction of hTERT mRNA in proliferating lymphocytes derived from five of seven patients was lower than that in unaffected control individuals (P<.05). The patient lymphocytes exhibited shorter telomeres than age-matched unaffected individuals (P<.0001). A reduction in replicative life span and a high rate of chromosome fusions were observed in cultured patient fibroblasts. Reconstitution of telomerase activity by ectopic expression of hTERT extended the telomere length, increased the population doublings, and prevented the end-to-end fusion of chromosomes. We conclude that hTERT is limiting and haploinsufficient for telomere maintenance in humans in vivo. Accordingly, the hTERT deletion may be one genetic element contributing to the phenotypic changes in CdCS.

[Paternity assignment of giant panda by microsatellite genotyping].

We used genotype data across 18 microsatellite loci to establish the paternity of giant panda cubs at the Chengdu Zoo and the Chengdu Research Base of Giant Panda Breeding. The results demonstrate that the combined exclusion probability using these 18 microsatellite loci is 0.999921 while confidence is 95 % when the mother is known; if mother is unknown then the exclusion probability is 0.994109, but the confidence of this is less than 80%. Since the mother-offspring relationship is known in captive populations, the results could resolve unknown paternities in the Chengdu ex situ populations. However, to establish accurately the genetic relationships of wild giant pandas,more microsatellite loci may be required.

Genetic alterations in cervical carcinomas: frequent low-level amplifications of oncogenes are associated with human papillomavirus infection.

The development of cervical carcinoma is closely associated with HPV infection. However, other genetic alterations also play an important role. In this study, we analyzed copy number alterations of several oncogene loci in a panel of 84 cervical tumors. Sixty-five (77%) tumors were HPV DNA-positive, and most were infected with type 16 or type 18 or both. The oncogenes studied include PIK3CA at 3q26.3, TERT at 5p15.33, C-MYC at 8q24, CCND1 at 11q13.3, ERBB2 at 17q21.2 and locus region 20q13.2. Amplification of 1 or more genes was detected in 55 (65%) cases using interphase FISH. PIK3CA was amplified in 43% of tumors, followed by TERT (33%), 20q13.2 (30%), ERBB2 (29%), C-MYC (25%) and CCND1 (12%). Most tumors showed low-level amplification with 3-7 copies of these genes, and complex changes involving 3 or more genes occur more frequently in tumors at advanced stages. Increased protein expression of c-erbB2 and c-myc was observed in tumors with the corresponding gene amplification. Oncogene alterations were found more often in HPV-infected cases, particularly for C-MYC and TERT. These findings indicate that HPV-associated cervical carcinomas bear frequent alterations of these genes, which may have critical biologic impact on the development and progression of carcinoma of the uterine cervix.

Co-existence of SYT-SSX1 and SYT-SSX2 fusions in synovial sarcomas.

The chromosomal translocation t(X;18)(p11.2;q11.2) is tightly linked to the tumorigenesis of synovial sarcoma. Through this translation the SYT gene on chromosome 18 is fused with a testis/cancer antigen gene on the X chromosome, generating either a SYT-SSX1, SYT-SSX2, or less often a SYT-SSX4 fusion gene. It has been anticipated that the individual synovial sarcoma carries only one of these variants, however, in this study we demonstrated that SYT-SSX1 and SYT-SSX2 co-exist in a significant proportion of the cases. From 121 SYT-SSX positive primary tumors, co-expression of SYT-SSX1 and SYT-SSX2 was seen in 12 cases (10%), which were characterized in further detail both at the RNA, DNA and chromosomal level. In all 12 cases the SYT-SSX1 and SYT-SSX2 fusions resulted in identical SYT-SSX fusion transcripts. However, at the genomic level the translocations were different, and most likely occurred between variable intronic sites in the target genes. By interphase FISH analyses of 10 cases SYT-SSX2 translocations were found to be the most abundant in all but one of the cases, in which SYT-SSX1 was predominating. The findings reveal a new heterogenous feature of synovial sarcoma, accounting for approximately 10% of all cases, which may shed light on the molecular genetic mechanisms behind translocations in general, and on the etiology of synovial sarcoma in particular.

[Pedigree analysis of captive giant panda].

Pedigree analysis of captive giant panda was conducted by Sparks Ver1.4 Software. The result shows that genetic drift has a strong effect on the loss of genetic diversity. And the dispersal of captive giant panda is an acute dangerous factor when all of these small populations would be declined if inbreeding happened. For this reason, all these small populations should be managed as a whole unit to approach the goal for long-term conservation.

Amplification of the telomerase reverse transcriptase (hTERT) gene in cervical carcinomas.

The expression of telomerase reverse transcriptase (hTERT), the catalytic component of the telomerase complex, is required for activation of telomerase during immortalization and transformation of human cells. However, the biochemical and genetic mechanisms governing hTERT expression remain to be elucidated. In the present study, we examined hTERT amplification as a potential genetic event contributing to telomerase activation in cervical carcinomas. An amplification of the hTERT gene was found in 1/4 cervical cancer cell lines and 21/88 primary tumor samples derived from the patients with cervical carcinomas. An increase in the hTERT copy number was significantly correlated with higher levels of hTERT protein expression. Moreover, the hTERT alterations with the enhanced hTERT expression were exclusively observed in those tumors with high-risk human papillomavirus infection. Taken together, the hTERT gene amplification, directly or indirectly targeted by human papillomavirus, may be one of the driving forces responsible for upregulation of hTERT expression and activation of telomerase in cervical cancers.

The histone deacetylase inhibitor trichostatin A derepresses the telomerase reverse transcriptase (hTERT) gene in human cells.

Activation of telomerase, essential for cellular immortalization and transformation, requires the induction of its catalytic component, telomerase reverse transcriptase (hTERT). However, biochemical and genetic mechanisms for the control of hTERT expression remain undefined. In the present study, we demonstrate that the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) induces hyperacetylation of histones at the hTERT proximal promoter, directly transactivates the hTERT gene in normal human telomerase-negative cells, and upregulates hTERT expression in telomerase-positive tumor cells. Overexpression of HDAC1 leads to repression of the hTERT promoter activity. TSA-mediated activation of the hTERT promoter is abolished by the mutation of Sp1 sites at the proximal promoter, suggesting that the effect of TSA is regulated through Sp1 motifs. We also show a physical interaction of Sp1 with HDAC1 and the presence of HDAC1 at the hTERT promoter region. Moreover, hyperacetylation of histones at the hTERT promoter is associated with the natural up-regulation of hTERT expression that occurs in activated T lymphocytes. Taken together, histone acetylation/deacetylation may be a common underlying feature to hTERT transactivation/repression in human normal and malignant cells.