Article
RNA levels of human retrovirus receptors Pit1 and Pit2 do not correlate with infectibility by three retroviral vector pseudotypes.
Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.
Human Gene Therapy (impact factor:
4.22).
12/1998;
9(17):2619-27.
DOI:10.1089/hum.1998.9.17-2619
pp.2619-27
Source: PubMed
-
Citations (0)
- Cited In (5)
-
Article: Transduction of human primitive repopulating hematopoietic cells with lentiviral vectors pseudotyped with various envelope proteins.
[show abstract] [hide abstract]
ABSTRACT: Lentiviral vectors are useful for transducing primitive hematopoietic cells. We examined four envelope proteins for their ability to mediate lentiviral transduction of mobilized human CD34(+) peripheral blood cells. Lentiviral particles encoding green fluorescent protein (GFP) were pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G), the amphotropic (AMPHO) murine leukemia virus envelope protein, the endogenous feline leukemia viral envelope protein or the feline leukemia virus type C envelope protein. Because the relative amount of genome RNA per ml was similar for each pseudotype, we transduced CD34(+) cells with a fixed volume of each vector preparation. Following an overnight transduction, CD34(+) cells were transplanted into immunodeficient mice which were sacrificed 12 weeks later. The average percentages of engrafted human CD45(+) cells in total bone marrow were comparable to that of the control, mock-transduced group (37-45%). Lenti-particles pseudotyped with the VSV-G envelope protein transduced engrafting cells two- to tenfold better than particles pseudotyped with any of the gamma-retroviral envelope proteins. There was no correlation between receptor mRNA levels for the gamma-retroviral vectors and transduction efficiency of primitive hematopoietic cells. These results support the use of the VSV-G envelope protein for the development of lentiviral producer cell lines for manufacture of clinical-grade vector.Molecular Therapy 04/2010; 18(7):1310-7. · 6.87 Impact Factor -
Article: Mapping of the minimal inorganic phosphate transporting unit of human PiT2 suggests a structure universal to PiT-related proteins from all kingdoms of life.
[show abstract] [hide abstract]
ABSTRACT: The inorganic (Pi) phosphate transporter (PiT) family comprises known and putative Na(+)- or H(+)-dependent Pi-transporting proteins with representatives from all kingdoms. The mammalian members are placed in the outer cell membranes and suggested to supply cells with Pi to maintain house-keeping functions. Alignment of protein sequences representing PiT family members from all kingdoms reveals the presence of conserved amino acids and that bacterial phosphate permeases and putative phosphate permeases from archaea lack substantial parts of the protein sequence when compared to the mammalian PiT family members. Besides being Na(+)-dependent P(i) (NaP(i)) transporters, the mammalian PiT paralogs, PiT1 and PiT2, also are receptors for gamma-retroviruses. We have here exploited the dual-function of PiT1 and PiT2 to study the structure-function relationship of PiT proteins. We show that the human PiT2 histidine, H(502), and the human PiT1 glutamate, E(70),--both conserved in eukaryotic PiT family members--are critical for P(i) transport function. Noticeably, human PiT2 H(502) is located in the C-terminal PiT family signature sequence, and human PiT1 E(70) is located in ProDom domains characteristic for all PiT family members.A human PiT2 truncation mutant, which consists of the predicted 10 transmembrane (TM) domain backbone without a large intracellular domain (human PiT2ΔR(254)-V(483)), was found to be a fully functional P(i) transporter. Further truncation of the human PiT2 protein by additional removal of two predicted TM domains together with the large intracellular domain created a mutant that resembles a bacterial phosphate permease and an archaeal putative phosphate permease. This human PiT2 truncation mutant (human PiT2ΔL(183)-V(483)) did also support P(i) transport albeit at very low levels. The results suggest that the overall structure of the P(i)-transporting unit of the PiT family proteins has remained unchanged during evolution. Moreover, in combination, our studies of the gene structure of the human PiT1 and PiT2 genes (SLC20A1 and SLC20A2, respectively) and alignment of protein sequences of PiT family members from all kingdoms, along with the studies of the dual functions of the human PiT paralogs show that these proteins are excellent as models for studying the evolution of a protein's structure-function relationship.BMC Biochemistry 05/2011; 12:21. · 1.99 Impact Factor -
Article: Regulation of cell proliferation and cell density by the inorganic phosphate transporter PiT1.
[show abstract] [hide abstract]
ABSTRACT: ABSTACT: The inorganic phosphate (Pi) transporter, PiT1 (SLC20A1), is ubiquitously expressed in mammalian cells. It has previously been shown that down-regulation of PiT1 severely impaired the proliferation of two transformed human cells lines, HepG2 and HeLa, and the tumorigenicity of HeLa cells in nude mice. Moreover, PiT1 knock-out mice do not survive past E12.5 and from E10.5, the embryos were found to be growth-retarded and showed reduced proliferation of liver cells. Isolated mouse embryonic fibroblasts with knocked out as well as reduced PiT1 expression levels also exhibited impaired proliferation. Together these results suggest that a certain level of PiT1 is important for proliferation. We have here investigated the role of PiT1 in regulation of cell proliferation using two strictly density-inhibited cells lines, the murine MC3T3-E1 and NIH3T3 cells. We found that knock-down of PiT1 in MC3T3-E1 cells led to impaired proliferation supporting that at least a certain level of PiT1 is important for wildtype level of proliferation. We, however, also observed that MC3T3-E1 and NIH3T3 cells themselves regulate their endogenous PiT1 mRNA levels with lower levels in general correlating with decreased proliferation/increased cell density. Moreover, over-expression of human PiT1 led to increased proliferation of both MC3T3-E1 and NIH3T3 cultures and resulted in higher cell densities in cultures of these two strictly density-inhibited cell lines. In addition, when we transformed NIH3T3 cells by cultivation in fetal bovine serum, cells over-expressing human PiT1 formed more colonies in soft agar than control cells. We conclude that not only is a certain level of PiT1 necessary for normal cell division as suggested by previously published studies, rather the cellular PiT1 level is involved in regulating cell proliferation and cell density and an increased PiT1 expression can indeed make NIH3T3 cells more sensitive to transformation. We have thus provided the first evidence for that expression of the type III Pi transporter, PiT1, above the endogenous level can drive cell proliferation and overrule cell density constraints, and the results bridge previous observations showing that a certain PiT1 level is important for regulating normal embryonic growth/development and for tumorigenicity of HeLa cells.Cell Division 03/2012; 7(1):7. · 3.00 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed.
The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual
current impact factor.
Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence
agreement may be applicable.
Keywords
14 human cell lines
A-MuLV pseudotyped vectors
A-MuLV pseudotypes
amphotropic murine leukemia virus
cell lines
GaLV pseudotypes
gibbon ape leukemia virus
higher affinity
human cells
human tissues
mRNA levels
Northern analysis
partial reciprocal interference
Pit1 RNA
Pit1-specific mRNA
Pit2 mRNA
Pit2 mRNA levels
Pit2 RNA
Pit2 RNA levels
specific receptors
