Lncap
Encyclopedia
LNCaP cells are a cell line of human cells commonly used in the field of oncology
. LNCaP cells are androgen
-sensitive human prostate
adenocarcinoma
cells derived from the left supraclavicular lymph node metastasis
from a 50-year-old caucasian male in 1977. They are adherent epithelial cells growing in aggregates and as single cells.
One major obstacle to the conducting the most clinically relevant Prostate Cancer
(PCa) research has been the lack of cell lines that closely mimic human disease progression. Two hallmarks of metastatic human prostate cancer include the shift of aggressive PCa from androgen-sensitivity to an Androgen Insensitive (AI) state, and the propensity of PCa to metastasize to bone. Although the generation of AI cell lines has been quite successful as demonstrated in the “classic” cell lines DU145
and PC3
, the behavior of these cells in bone does not fully mimic clinical human disease. It is well established that human PCa bone metastasis form osteoblastic lesions rather than osteolytic lesions seen in other cancers like breast cancer. Similarly, PC-3 and DU145 cells form osteolytic tumors. To develop an AI-PCa cell model that more closely mimics clinical disease, LNCaP sublines have been generated to provide the most clinically relevant tissue culture tools to date.
. LNCaP cells have a modal chromosome number of 76 to 91, indicative of a human male karyotype with several marker chromosome
s. The malignant properties of LNCaP cells are maintained in athymic nude mice which develop tumors at the injection site and show a similar doubling time in vivo.
High-affinity specific androgen
and estrogen
receptors are present in the cytosol and nuclear fractions. The LNCaP line is hormonally
responsive, shown by in vitro 5 alpha-dihydrotestosterone modulation of cell growth and acid phosphatase production. LNCaP cells also express Prostate Specific Antigen
([PSA]). In vivo, Male mice develop tumors earlier and at a greater frequency than do females and hormonal manipulations show that the frequency of tumor development correlates with serum androgen levels. The rate of the tumor growth, however, is independent of the gender or hormonal status of the host..
To replicate the hallmark shift of PCa cells to AI, LNCaP host mice were castrated by way of midscrotal incision at approximately 8 weeks post injection. Tumors were maintained in castrated hosts for 4 to 5 weeks at which time remaining tumors were harvested. In total, two subsets of cells were collected from castrated hosts: C4 and C5, collected at 4 and 5 weeks respectively.
To further select for AI-PCa cells, the C4 subline was co-injected with MS human fibroblasts into a castrated host. The resulting tumors were isolated and an additional subline was generated, C4-2.
Karotype comparisons indicate that
LNCaP cells grown in intact hosts (M subline) have a modal chromosomal distribution number of 83, C4 and C5 sublines with 85, and the C4-2 subline with 83.
To further verify that these cells were of human origin karotype analysis determined that the parental LNCaP cells had 7 distinct marker chromosomes, with two copies of each. The M, C4, C5, and C4-2 sublines contained most of the marker chromosomes, with the M subline being most similar to the parental LNCaP cells. C4,C5 and C4-2 are only minutely distinct from LNCaP and the M subline with the addition of a marker chromosome resulting from a segment addition to chromosome 6. Interestingly, a Y chromosome
is not present in most C4, C5 and C4-2 cells, suggesting major chromosomal alterations.
C4,C5, and C4-2 sublines grow well under identical tissue culture conditions as LNCaP with similar growth rates. Parental LNCaP, M, C4, and C5 subline have similar baseline gene expression levels of ornithine decarboxylase
(ODC) and Prostate Specific Antigen
(PSA) however, M, C4, and C5 sublines express 5-10X more PSA mRNA. M, C4, C5 and C4-2 also expressed reduced human androgen receptor
mRNA as expected in AI cells.
Androgen Insensitivity
All sublines were treated with dihydrotestosterone
(DHT), a high-affinity ligand for AR. DHT treatment elicited markedly reduced growth in C4 and C5 cells and no growth in C4-2 cells when compared to the high rate of growth seen in LNCaP cells, suggesting reduced androgen sensitivity in C4 and C5 and AI in C4-2 cells. Whole-cell AR assay also indicated that LNCaP cells have a much higher affinity form of AR (Kd = 159 pM) when compared to C4-2 (Kd = 267 pM).
Tumorigenicity
C4 and C5 sublines exhibit greatly increased tumorigenicity when injected in intact male mice, unlike parental LNCaP cells. C4 and C5 were also able to form highly vascularized carcinomas in castrated mice when co-injected with MS human fibroblasts. The C4-2 subline more readily forms tumors in intact hosts that C4 and C5 sublines and interestingly, are the only cells able to form tumors in castrated host without co-injection with MS human bone fibroblasts. These same C4-2 tumors stained for PSA and secreted high levels of PSA into the growth medium.
C4-2B cells were positive for PSA and cytokeratin 8, confirming their prostatic origin. Most importantly, immunohistochemical staining of the C4-2B tumors were positive for osteoblast activity suggesting the induction of osteoblastic tumor formation mirroring the progression of human PCa.
When cultured in a “promineralization medium” that contains ascorbic acid
(known to promote skeletal-like ECM formation in osteoblasts) and a source of phosphate (for hydroxyapatite formation), C4-2B cells produce and retain approximately 8x more mineralized calcium than parental LNCaP cells. C4-2B cells also express higher levels of osteoprotegerin
(OPN), alkaline phosphatase
, bone sialoprotein
(BSP), Osteocalcin
(OCN), RANKL
, and Osteonectin
(OSN) mRNA, all of which are highly expressed by osteoblasts.
Osteoblast promoter activity is also higher in C4-2B cells when compared to LNCaP, as indicated by Cbfa1 transcription factor expression. Concomitantly, BMP7, a known inducer of Cbaf1, is also more highly expressed in C4-2B cells, further suggesting many osteoblast-like properties.
Oncology
Oncology is a branch of medicine that deals with cancer...
. LNCaP cells are androgen
Androgen
Androgen, also called androgenic hormone or testoid, is the generic term for any natural or synthetic compound, usually a steroid hormone, that stimulates or controls the development and maintenance of male characteristics in vertebrates by binding to androgen receptors...
-sensitive human prostate
Prostate
The prostate is a compound tubuloalveolar exocrine gland of the male reproductive system in most mammals....
adenocarcinoma
Adenocarcinoma
Adenocarcinoma is a cancer of an epithelium that originates in glandular tissue. Epithelial tissue includes, but is not limited to, the surface layer of skin, glands and a variety of other tissue that lines the cavities and organs of the body. Epithelium can be derived embryologically from...
cells derived from the left supraclavicular lymph node metastasis
Metastasis
Metastasis, or metastatic disease , is the spread of a disease from one organ or part to another non-adjacent organ or part. It was previously thought that only malignant tumor cells and infections have the capacity to metastasize; however, this is being reconsidered due to new research...
from a 50-year-old caucasian male in 1977. They are adherent epithelial cells growing in aggregates and as single cells.
One major obstacle to the conducting the most clinically relevant Prostate Cancer
Prostate cancer
Prostate cancer is a form of cancer that develops in the prostate, a gland in the male reproductive system. Most prostate cancers are slow growing; however, there are cases of aggressive prostate cancers. The cancer cells may metastasize from the prostate to other parts of the body, particularly...
(PCa) research has been the lack of cell lines that closely mimic human disease progression. Two hallmarks of metastatic human prostate cancer include the shift of aggressive PCa from androgen-sensitivity to an Androgen Insensitive (AI) state, and the propensity of PCa to metastasize to bone. Although the generation of AI cell lines has been quite successful as demonstrated in the “classic” cell lines DU145
Du145
DU145 and PC3 human prostate cancer cell lines are the "classical" cell lines of prostatic cancer. DU145 cells have moderate metastatic potential compared to PC3 cells which have high metastatic potential....
and PC3
Pc3
PC3 and DU145 human prostate cancer cell lines are the "classical" cell lines of prostatic cancer. PC3 cells have high metastatic potential compared to DU145 cells which have a moderate metastatic potential....
, the behavior of these cells in bone does not fully mimic clinical human disease. It is well established that human PCa bone metastasis form osteoblastic lesions rather than osteolytic lesions seen in other cancers like breast cancer. Similarly, PC-3 and DU145 cells form osteolytic tumors. To develop an AI-PCa cell model that more closely mimics clinical disease, LNCaP sublines have been generated to provide the most clinically relevant tissue culture tools to date.
History
The LNCaP cell line was established from a metastatic lesion of human prostatic adenocarcinoma. The LNCaP cells grow readily in vitro (up to 8 x 105 cells/sq cm; doubling time, 60 hr), form clones, are highly resistant to human fibroblast interferonInterferon
Interferons are proteins made and released by host cells in response to the presence of pathogens—such as viruses, bacteria, or parasites—or tumor cells. They allow communication between cells to trigger the protective defenses of the immune system that eradicate pathogens or tumors.IFNs belong to...
. LNCaP cells have a modal chromosome number of 76 to 91, indicative of a human male karyotype with several marker chromosome
Chromosome
A chromosome is an organized structure of DNA and protein found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.Chromosomes...
s. The malignant properties of LNCaP cells are maintained in athymic nude mice which develop tumors at the injection site and show a similar doubling time in vivo.
High-affinity specific androgen
Androgen
Androgen, also called androgenic hormone or testoid, is the generic term for any natural or synthetic compound, usually a steroid hormone, that stimulates or controls the development and maintenance of male characteristics in vertebrates by binding to androgen receptors...
and estrogen
Estrogen
Estrogens , oestrogens , or œstrogens, are a group of compounds named for their importance in the estrous cycle of humans and other animals. They are the primary female sex hormones. Natural estrogens are steroid hormones, while some synthetic ones are non-steroidal...
receptors are present in the cytosol and nuclear fractions. The LNCaP line is hormonally
Hormone
A hormone is a chemical released by a cell or a gland in one part of the body that sends out messages that affect cells in other parts of the organism. Only a small amount of hormone is required to alter cell metabolism. In essence, it is a chemical messenger that transports a signal from one...
responsive, shown by in vitro 5 alpha-dihydrotestosterone modulation of cell growth and acid phosphatase production. LNCaP cells also express Prostate Specific Antigen
Prostate specific antigen
Prostate-specific antigen also known as gamma-seminoprotein or kallikrein-3 is a glycoprotein that in humans is encoded by the KLK3 gene. KLK3 is a member of the kallikrein-related peptidase family that are secreted by the epithelial cells of the prostate gland...
([PSA]). In vivo, Male mice develop tumors earlier and at a greater frequency than do females and hormonal manipulations show that the frequency of tumor development correlates with serum androgen levels. The rate of the tumor growth, however, is independent of the gender or hormonal status of the host..
C4/C5 and C4-2
Wu et al. (1994) reproduced the human-derived LNCaP tumors in immunocompromised mice by co-injection of LNCaP cells with MS human bone fibroblasts. Cells were subcutaneously injected at multiple sites into the mouse flank and after approximately 4 weeks of growth, tumors were easily detectable by physical examination and had a high rate of growth (17-33 mm3/day).To replicate the hallmark shift of PCa cells to AI, LNCaP host mice were castrated by way of midscrotal incision at approximately 8 weeks post injection. Tumors were maintained in castrated hosts for 4 to 5 weeks at which time remaining tumors were harvested. In total, two subsets of cells were collected from castrated hosts: C4 and C5, collected at 4 and 5 weeks respectively.
To further select for AI-PCa cells, the C4 subline was co-injected with MS human fibroblasts into a castrated host. The resulting tumors were isolated and an additional subline was generated, C4-2.
Karotype comparisons indicate that
LNCaP cells grown in intact hosts (M subline) have a modal chromosomal distribution number of 83, C4 and C5 sublines with 85, and the C4-2 subline with 83.
To further verify that these cells were of human origin karotype analysis determined that the parental LNCaP cells had 7 distinct marker chromosomes, with two copies of each. The M, C4, C5, and C4-2 sublines contained most of the marker chromosomes, with the M subline being most similar to the parental LNCaP cells. C4,C5 and C4-2 are only minutely distinct from LNCaP and the M subline with the addition of a marker chromosome resulting from a segment addition to chromosome 6. Interestingly, a Y chromosome
Y chromosome
The Y chromosome is one of the two sex-determining chromosomes in most mammals, including humans. In mammals, it contains the gene SRY, which triggers testis development if present. The human Y chromosome is composed of about 60 million base pairs...
is not present in most C4, C5 and C4-2 cells, suggesting major chromosomal alterations.
C4,C5, and C4-2 sublines grow well under identical tissue culture conditions as LNCaP with similar growth rates. Parental LNCaP, M, C4, and C5 subline have similar baseline gene expression levels of ornithine decarboxylase
Ornithine decarboxylase
The enzyme ornithine decarboxylase catalyzes the decarboxylation of ornithine to form putrescine. This reaction is the committed step in polyamine synthesis. In humans, this protein has 461 amino acids and forms a homodimer....
(ODC) and Prostate Specific Antigen
Prostate specific antigen
Prostate-specific antigen also known as gamma-seminoprotein or kallikrein-3 is a glycoprotein that in humans is encoded by the KLK3 gene. KLK3 is a member of the kallikrein-related peptidase family that are secreted by the epithelial cells of the prostate gland...
(PSA) however, M, C4, and C5 sublines express 5-10X more PSA mRNA. M, C4, C5 and C4-2 also expressed reduced human androgen receptor
Androgen receptor
The androgen receptor , also known as NR3C4 , is a type of nuclear receptor that is activated by binding of either of the androgenic hormones testosterone or dihydrotestosterone in the cytoplasm and then translocating into the nucleus...
mRNA as expected in AI cells.
Androgen Insensitivity
All sublines were treated with dihydrotestosterone
Dihydrotestosterone
Dihydrotestosterone is an androgen or male sex hormone. The enzyme 5α-reductase synthesises DHT in the prostate, testes, hair follicles, and adrenal glands...
(DHT), a high-affinity ligand for AR. DHT treatment elicited markedly reduced growth in C4 and C5 cells and no growth in C4-2 cells when compared to the high rate of growth seen in LNCaP cells, suggesting reduced androgen sensitivity in C4 and C5 and AI in C4-2 cells. Whole-cell AR assay also indicated that LNCaP cells have a much higher affinity form of AR (Kd = 159 pM) when compared to C4-2 (Kd = 267 pM).
Tumorigenicity
C4 and C5 sublines exhibit greatly increased tumorigenicity when injected in intact male mice, unlike parental LNCaP cells. C4 and C5 were also able to form highly vascularized carcinomas in castrated mice when co-injected with MS human fibroblasts. The C4-2 subline more readily forms tumors in intact hosts that C4 and C5 sublines and interestingly, are the only cells able to form tumors in castrated host without co-injection with MS human bone fibroblasts. These same C4-2 tumors stained for PSA and secreted high levels of PSA into the growth medium.
C4-2B
To generate a bone metastatic subline, C4-2 cells were orthotopically injected into castrated male mice. These cells formed large primary tumors of the prostate, lymph nodes, as well as osseus tumors. Isolation of these osseus tumors resulted in the C4-2B subline.C4-2B cells were positive for PSA and cytokeratin 8, confirming their prostatic origin. Most importantly, immunohistochemical staining of the C4-2B tumors were positive for osteoblast activity suggesting the induction of osteoblastic tumor formation mirroring the progression of human PCa.
When cultured in a “promineralization medium” that contains ascorbic acid
Ascorbic acid
Ascorbic acid is a naturally occurring organic compound with antioxidant properties. It is a white solid, but impure samples can appear yellowish. It dissolves well in water to give mildly acidic solutions. Ascorbic acid is one form of vitamin C. The name is derived from a- and scorbutus , the...
(known to promote skeletal-like ECM formation in osteoblasts) and a source of phosphate (for hydroxyapatite formation), C4-2B cells produce and retain approximately 8x more mineralized calcium than parental LNCaP cells. C4-2B cells also express higher levels of osteoprotegerin
Osteoprotegerin
Osteoprotegerin , also known as osteoclastogenesis inhibitory factor , or tumor necrosis factor receptor superfamily member 11B , is a protein that in humans is encoded by the TNFRSF11B gene...
(OPN), alkaline phosphatase
Alkaline phosphatase
Alkaline phosphatase is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation...
, bone sialoprotein
Bone sialoprotein
Bone sialoprotein is a component of mineralized tissues such as bone, dentin, cementum and calcified cartilage. BSP is a significant component of the bone extracellular matrix and has been suggested to constitute approximately 8% of all non-collagenous proteins found in bone and cementum...
(BSP), Osteocalcin
Osteocalcin
Osteocalcin, also known as bone gamma-carboxyglutamic acid-containing protein , is a noncollagenous protein found in bone and dentin. In humans, the osteocalcin is encoded by the BGLAP gene.- Function :...
(OCN), RANKL
RANKL
Receptor activator of nuclear factor kappa-B ligand , also known as tumor necrosis factor ligand superfamily member 11 , TNF-related activation-induced cytokine , osteoprotegerin ligand , and osteoclast differentiation factor , is a protein that in humans is encoded by the TNFSF11 gene.RANKL is...
, and Osteonectin
Osteonectin
Osteonectin also known as secreted protein acidic and rich in cysteine or basement-membrane protein 40 is a protein that in humans is encoded by the SPARC gene....
(OSN) mRNA, all of which are highly expressed by osteoblasts.
Osteoblast promoter activity is also higher in C4-2B cells when compared to LNCaP, as indicated by Cbfa1 transcription factor expression. Concomitantly, BMP7, a known inducer of Cbaf1, is also more highly expressed in C4-2B cells, further suggesting many osteoblast-like properties.