Cell Immortalization 

Recall that Weinberg ‘sprinkled’ pieces of DNA from a human bladder cancer onto mouse fibroblasts in order to find (and later identify) the first human oncogene Ras.
Evidence of this transformation event was seen as a focus of cells in a culture dish that had seemingly acquired the ability to divide without end.

In this context, one can imagine the focus as a colony of identical cells, or clones; i.e., that these ‘tumors in a dish’ were monoclonal in nature

Lineage tracing using unique biomarkers can identify the monoclonal origins of cancer.

Leiomyomas, or more commonly uterine fibroids, are benign growths of the muscular lining of the uterine wall. Here, scientists have taken advantage of the location of the glucose-6-phosphate dehydrogenase (G6PD) gene on the X chromosome and the occasional sensitivity of the enzyme to heat to demonstrate the monoclonality of these fibroids, and of cancer in general.

Since one X chromosome is often randomly inactivated (transcriptionally repressed) in the early embryo, and regresses into a small Barr body, adult women will have patches within their own bodies where either the maternal or paternal G6PD allele predominates. The same will thus be true of fibroids. When women are heterozygous at the G6PD locus, this is recognized in individual fibroids by the presence of either a heat sensitive or insensitive G6PD allele.

Cancer cells retain some of the properties of ‘their youth’ thereby providing evidence of their clonality.

The monoclonality of cancer can be used to make predictions about the number of cell divisions (population doublings) to make recognizable tumors

Defense mechanisms are often deployed that make the actual number of required population doublings to form large tumors far higher than these estimates.

Innate barriers (pRb, p53) to cell division are employed as cells recognize their own cell growth misbehaviors.
Tumor growth becomes limited by poor access to nutrients and oxygen and the inability to eliminate metabolic wastes; i.e., large tumors may be poorly vascularized.

Primary cultured cells go through about 60 populations doublings which is not enough to make a tumor

Based on the marker enzyme β-galactosidase, one can visualize senescence in cultured cells, in aging tissues, and in tissues exposed to hypoxia, ROS, radiation, and chemotherapeutic drugs.

The replicative senescence of cells seems to arise, in part, from the increased expression of two CDK inhibitors p16INK4A and p21Cip1.
Ectopic expression of p16INK4A in fibroblasts can arbitrarily create the telltale signs of a senescent fibroblast long before ‘the end of its natural prime’.

The replicative senescence of cells seems to arise, in part, from the increased expression of two CDK inhibitors p16INK4A and p21Cip1.
Changing to a special media and/or a special type of plastic used in the culture dish can repress expression of p16INK4A and p53 (and therefore p21Cip1), and therefore repress the senescence of cultured cells.

Senescence is therefore something cells can sidestep.

Cells in culture can also overcome senescence through the expression of the SV40 large T antigen (LT), an oncoprotein that can bind and sequester pRb and p53

Beside senescence, cells must also deal with crisis, the wide-spread apoptosis of cells.

Senescence and crisis are based on a hard-wired counting device for population doublings in cells found in the telomeres of chromosomal DNA.

Telomeres prevent end joining of chromosomes, as does the TRF2 protein. Lack of TRF2 generates long, continuous, and therefore problematic chromosomes.

Telomeres are made up of repeated hexanucleotide sequences (5’-TTAGGG-3’) that extend for 5-10kb in length at the ends of chromosomes.

Telomere architecture is designed for DNA protection.

• The 3’ G-rich strand exceeds the 5’ C-rich strand by several hundred nucleotides.
• This 3’ G-rich strand invades into and displaces a portion of its upstream sequence in order to hybridize with the 5’ C-rich strand (see D-loop and t-loop).

Telomeres are also protected and stabilized by proteins of the shelterin complex.

Telomeres are lost during successive rounds of DNA synthesis. This occurs because the RNA primers (circled) needed for lagging strand synthesis are only laid down hundreds of nucleotides apart and likely do not catch the very end of the telomeres. Also, exonucleases cause loss of DNA from telomeres. Collectively, this accounts for 50-100bp of nucleotide loss per cellular generation.

Telomere erosion can be measured with each cell division.
Crisis (and, in contrast, the immortalization of cancer cells) are therefore held in the balance of telomere length.

Radioactive probes recognize ever decreasing telomere restriction fragments (TRF) from the ends of chromosomes in a population of lymphocytes of increasing passage number. These cells eventually enter crisis and die, though some may immerge on the other end immortalized where telomere length stabilizes.

Loss of telomeres leads to end-joining of chromosomes and catastrophic alterations in chromosome number and distribution (dicentric chromosomes) in daughter cells following mitosis
These so-called breakage-fusion-bridge cycles can quickly lead to apoptosis, even in the absence of p53.

So given the limitations in growth faced by primary cells, how then do cancer cells become immortalized? Cancer cells overexpress telomerase. Nearly 9 out of 10 cancers have telomerase activity which arises out of crisis as a re-expression of genes that were once present in early embryogenesis but silenced somewhere during cell differentiation

The telomerase holoenzyme uses an RNA molecule (hTR) to repeatedly template DNA synthesis to the 3’ end of telomeres through reverse transcriptase activity (hTERT). A separate DNA polymerase finishes the job.

Ectopic expression of hTERT can block entry into crisis altogether and lead directly to cell line immortalization.

So what do you think the phenotype would be of an mTR-deficient mouse? Nothing really different from a wild-type mouse in the first generation. However, the mice become sickly by the 5th and 6th generations because loss of telomeres eventually compromised chromosomal integrity leading to apoptosis in dividing cells and atrophy in organ tissue

So what do you think the phenotype would be of an mTR-deficient mouse in the absence of p53? Mice become more cancer prone with each generation because Loss of telomeres compromises chromosomal integrity, but apoptosis does not occur. Cells that survive reactivate telomere formation via an alternative pathway.

For those small numbers of tumors that don’t express telomerase, there is an alternative (ALT) method of telomere lengthening.

DNA duplication events between different chromosomes seems to contribute to the maintenance of telomeres in telomerase-negative, ALT-positive cancer cells. Oddly the ALT pathway is prevalent in osteosarcomas, other soft tissue sarcomas, and glioblastomas.

Cells have a finite replicative potential.

Senescent cells are permanently incapable of entering the cell cycle (even though they are still metabolically active). Senescence can be related to physiological stress (e.g., oxidative damage).
Cells also count the number of cell divisions they go through. When that limit is reached, they enter ‘crisis’ and die by apoptosis.

Delayed acquisition of telomerase function may provide a mechanism for increased mutability early in tumorigenesis followed by stabilization of the mutant genome of cancer cells.